Nucleic acids encoding P-selectin ligand fusion proteins

ABSTRACT

A novel P-selectin ligand glycoprotein is disclosed, comprising the amino acid sequence set forth in SEQ ID NO:2 or by the amino acid sequence set forth in SEQ ID NO:4. DNA sequences encoding the P-selectin ligand protein are also disclosed, along with vectors, host cells, and methods of making the P-selectin ligand protein. Pharmaceutical compositions containing the P-selectin ligand protein and methods of treating inflammatory disease states characterized by P-selectin- and E-selectin-mediated intercellular adhesion are also disclosed.

This application is a division of application Ser. No. 09/935,144, filedAug. 21, 2001, now U.S. Pat. No. 7,563,760, which is a continuation ofapplication No. 08/713,556, filed Aug. 30, 1996, issued as U.S. Pat. No.6,277,975, which is a continuation-in-part of application No.08/428,734, filed Apr. 25, 1995, issued as U.S. Pat. No. 5,843,707,which is a continuation-in-part of application No. 08/316,305, filedSep. 30, 1994, now abandoned, which is a continuation-in-part ofapplication No. 08/235,398, filed Apr. 28, 1994, now abandoned, which isa continuation-in-part of application No. 08/112,608, filed Aug. 26,1993, now abandoned, which is a continuation-in-part of application No.07/965,662, filed Oct. 23, 1992, now abandoned, which are incorporatedby reference in their entireties. This application also claims priorityfrom International application No. PCT/US93/10168, filed Oct. 22, 1993,which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to the field of anti-inflammatorysubstances which act by inhibiting leukocyte adhesion to endothelialcells. More particularly, the present invention is directed to novelligands for the mammalian adhesion proteins known as selecting.

During inflammation leukocytes adhere to the vascular endothelium andenter subendothelial tissue, an interaction which is mediated byspecific binding of the selectin or LEC-CAM class of proteins to ligandson target cells. Such selectin-mediated cellular adhesion also occurs inthrombotic disorders and parasitic diseases and may be implicated inmetastatic spread of tumor cells.

The selectin proteins are characterized by a N-terminal lectin-likedomain, an epidermal growth factor-like domain, and regions of homologyto complement binding proteins. Thus far three human selectin proteinshave been identified, E-selectin (formerly ELAM-1), L-selectin (formerlyLAM-1) and P-selectin (formerly PADGEM or GMP-140). E-selectin isinduced on endothelial cells several hours after activation bycytokines, mediating the calcium-dependent interaction betweenneutrophils and the endothelium. L-selectin is the lymphocyte homingreceptor, and P-selectin rapidly appears on the cell surface ofplatelets when they are activated, mediating calcium-dependent adhesionof neutrophils or monocytes to platelets. P-selectin is also found inthe Weibel-Palade bodies of endothelial cells; upon its release fromthese vesicles P-selectin mediates early binding of neutrophils tohistamine- or thrombin-stimulated endothelium.

Selectins are believed to mediate adhesion through specific interactionswith ligands present on the surface of target cells. Generally theligands of selectins are comprised at least in part of a carbohydratemoiety. For example, E-selectin binds to carbohydrates having theterminal structure

and also to carbohydrates having the terminal structure

where R=the remainder of the carbohydrate chain. These carbohydrates areknown blood group antigens and are commonly referred to as sialylLewis^(x) and sialyl Lewis^(a), respectively. The presence of the sialylLewis^(x) antigen alone on the surface of an endothelial cell may besufficient to promote binding to an E-selectin expressing cell.E-selectin also binds to carbohydrates having the terminal structures

As with E-selectin, each selectin appears to bind to a range ofcarbohydrates with varying affinities. The strength of the selectinmediated adhesive event (binding affinity) may also depend on thedensity of the carbohydrate and on the density of the selectin on thecell surface.

P-selectin binds to carbohydrates containing the non-sialated form ofthe Lewis^(x) blood group antigen and with higher affinity to sialylLewis^(x). P-selectin may also recognize sulfatides, which areheterogeneous 3-sulfated galactosyl ceramides, isolated from myeloid andtumor cells by lipid extraction. However, the binding of cells bearingP-selectin to cells bearing P-selectin ligands is abolished when theligand-bearing cells are treated with proteases, indicating that theP-selectin ligand may be a glycoprotein.

Two putative glycoprotein ligands for P-selectin have recently beenidentified, one of which has been partially purified, (Moore et al., J.Cell Biol. 118, 445-456 (1992)). However, neither amino acid compositionnor the amino acid sequence of these glycoproteins are disclosed.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a compositioncomprising an isolated DNA encoding a P-selectin ligand protein, saidprotein comprising the amino acid sequence set forth in SEQ ID NO:2 fromamino acid 1 to amino acid 402. Also provided is a compositioncomprising an isolated DNA encoding a soluble P-selectin ligand protein,said protein comprising the amino acid sequence set forth in SEQ ID NO:2from amino acid 1 to amino acid 310. The invention further provides acomposition comprising an isolated DNA encoding a mature P-selectinligand protein, said protein comprising the amino acid sequence setforth in SEQ ID NO:2 from amino acid 42 to amino acid 402. In anotherembodiment, the invention provides a composition comprising an isolatedDNA encoding a soluble mature P-selectin ligand protein, said proteincomprising the amino acid sequence set forth in SEQ ID NO:2 from aminoacid 42 to amino acid 310. In another embodiment, the invention providesa composition comprising an isolated DNA encoding a P-selectin ligandprotein, said protein comprising the amino acid sequence set forth inSEQ ID NO:4. The invention further provides a composition comprising anexpression vector comprising any one of the isolated DNAs of theinvention, said DNA being operably linked to an expression controlsequence; a host cell transformed with the expression vector containingany one of the DNAs described above; and a process for producing theP-selectin ligand protein, which comprises:

(a) culturing a host cell transformed with an expression vectorcontaining any one of the DNAs of the invention in a suitable culturemedium; and

(b) purifying the P-selectin ligand protein from the culture medium.

In another embodiment, the invention provides a composition comprising aprotein comprising the amino acid sequence set forth in SEQ ID NO:2 fromamino acid 21 to amino acid 402, said protein being substantially freefrom other mammalian proteins. The invention further comprises a solubleP-selectin ligand protein comprising the amino acid sequence set forthin SEQ ID NO:2 from amino acid 21 to amino acid 310, said protein beingsubstantially free from other mammalian proteins. In another embodiment,the invention comprises a P-selectin ligand protein comprising the aminoacid sequence set forth in SEQ ID NO:2 from amino acid 1 to amino acid402, said protein being substantially free from other mammalianproteins. The invention also provides a composition comprising a matureP-selectin ligand protein comprising the amino acid sequence set forthin SEQ ID NO:2 from amino acid 42 to amino acid 402, said protein beingsubstantially free from other mammalian proteins. Further provided is acomposition comprising a soluble mature P-selectin ligand proteincomprising the amino acid sequence set forth in SEQ ID NO:2 from aminoacid 42 to amino acid 310, said protein being substantially free fromother mammalian proteins. In another embodiment, the invention providesa composition comprising a protein comprising the amino acid sequenceset forth in SEQ ID NO:4.

In yet another embodiment, the invention provides compositionscomprising antibodies specific for P-selectin ligand proteins.

In another embodiment, the invention provides a method of identifying aninhibitor of P-selectin-mediated intercellular adhesion which comprises:

(a) combining a P-selectin protein with a P-selectin ligand proteincomprising an amino acid sequence selected from the group consisting ofthe amino acid sequence set forth in SEQ ID NO:2 from amino acid 1 toamino acid 402, the amino acid sequence set forth in SEQ ID NO:2 fromamino acid 42 to amino acid 402, the amino acid sequence set forth inSEQ ID NO:2 from amino acid 42 to amino acid 310, and the amino acidsequence set forth in SEQ ID NO:4, said combination forming a firstbinding mixture;

(b) measuring the amount of binding between the P-selectin protein andthe P-selectin ligand protein in the first binding mixture;

(c) combining a compound with the P-selectin protein and the P-selectinligand protein to form a second binding mixture;

(d) measuring the amount of binding in the second binding mixture; and

(e) comparing the amount of binding in the first binding mixture withthe amount of binding in the second binding mixture;

wherein the compound is capable of inhibiting P-selectin-mediatedintercellular adhesion when a decrease in the amount of binding of thesecond binding mixture occurs.

In another embodiment, the invention provides a method of identifying aninhibitor of E-selectin-mediated intercellular adhesion which comprises:

(a) combining a E-selectin protein with a P-selectin ligand proteincomprising an amino acid sequence selected from the group consisting ofthe amino acid sequence set forth in SEQ ID NO:2 from amino acid 1 toamino acid 402, the amino acid sequence set forth in SEQ ID NO:2 fromamino acid 42 to amino acid 402, the amino acid sequence set forth inSEQ ID NO:2 from amino acid 42 to amino acid 310, and the amino acidsequence set forth in SEQ ID NO:4, said combination forming a firstbinding mixture;

(b) measuring the amount of binding between the E-selectin protein andthe P-selectin ligand protein in the first binding mixture;

(c) combining a compound with the E-selectin protein and the P-selectinligand protein to form a second binding mixture;

(d) measuring the amount of binding in the second binding mixture; and

(e) comparing the amount of binding in the first binding mixture withthe amount of binding in the second binding mixture;

wherein the compound is capable of inhibiting E-selectin-mediatedintercellular adhesion when a decrease in the amount of binding of thesecond binding mixture occurs. These methods may also be used to lookfor L-selectin inhibitors by substituting L-selectin for E-selectin.

The invention also encompasses processes for producing P-selectin ligandproteins which comprise (a) co-transforming a host cell with a DNAencoding a P-selectin ligand protein and a DNA encoding afucosyltransferase capable of synthesizing sialyl Lewis X (sLe^(x)) orsialyl Lewis A (sLe^(a)) (such as an (α1,3/α1,4) fucosyltransferase oran (α1,3) fucosyltransferase), each of said DNAs being operably linkedto an expression control sequence; (b) culturing the host cell insuitable culture medium; and (c) purifying the P-selectin ligand proteinfrom the culture medium. In certain other embodiments, the host cell isalso co-transformed with a DNA encoding a paired basic amino acidconverting enzyme and/or a DNA encoding a GlcNAc transferase (preferablya “core2 transferase”). In preferred embodiments, the P-selectin ligandprotein is a full-length or soluble form.

In other embodiments, the present invention includes a P-selectin ligandprotein having P-selectin ligand protein activity. In preferredembodiments, the ligand protein is a protein comprising the sequencefrom amino acid 42 to amino acid 60 of SEQ ID NO: 2, consistingessentially of the sequence from amino acid 42 to amino acid 60 of SEQID NO: 2, comprising the sequence from amino acid 42 to amino acid 88 ofSEQ ID NO: 2, consisting essentially of the sequence from amino acid 42to amino acid 88 of SEQ ID NO: 2, consisting essentially of the sequencefrom amino acid 42 to amino acid 118 of SEQ ID NO: 2, or consistingessentially of the sequence from amino acid 42 to amino acid 189 of SEQID NO: 2. In other preferred embodiments, at least one of the asparagineresidues at positions 65, 11 and 292 of SEQ ID NO: 2 have been deletedor replaced. Certain preferred embodiments of the ligand proteincomprises at least one of the tyrosine residues at positions 46, 48 and51 of SEQ ID NO: 2. DNAs encoding these P-selectin ligand proteins, hostcells transformed with such DNAs, process for producing protein byculturing such host cells, pharmaceutical compositions comprising theproteins, methods of identifying selectin binding inhibitors using theproteins, antibodies to the proteins and methods of inhibiting selectinmediated binding using the proteins are also encompassed by theinvention.

In yet other embodiments the invention provides an isolated DNA encodinga fusion protein comprising (a) a first amino acid sequence comprisingamino acid 42 to amino acid 60 of SEQ ID NO:2, and (b) a second aminoacid sequence derived from the sequence of a protein other thanP-selectin ligand. Preferably, an expression control sequence isoperably linked to the nucleotide sequence. Host cells transformed withsuch DNAs are also provided. The invention also provides a process forproducing a fusion protein, which comprises: (a) culturing the host cellunder condition suitable for expression of the fusion protein; and (b)purifying the fusion protein from the culture medium. Fusion proteinsproduced according to such process are also provide.

In certain preferred embodiments, the first amino acid sequence of suchfusion protein comprises amino acid 42 to amino acid 402 of SEQ ID NO:2,amino acid 42 to amino acid 310 of SEQ ID NO:2, amino acid 42 to aminoacid 88 of SEQ ID NO:2, amino acid 42 to amino acid 118 of SEQ ID NO:2,or amino acid 42 to amino acid 189 of SEQ ID NO:2.

In other preferred embodiments, the DNA comprises the nucleotidesequence of SEQ ID NO:35 from nucleotide 123 to nucleotide 939, thenucleotide sequence of SEQ ID NO:35, the nucleotide sequence of SEQ IDNO:37 from nucleotide 123 to nucleotide 807, the nucleotide sequence ofSEQ ID NO:37, the nucleotide sequence of SEQ ID NO:39 from nucleotide123 to nucleotide 1311, the nucleotide sequence of SEQ ID NO:39, thenucleotide sequence of SEQ ID NO:41 from nucleotide 123 to nucleotide792, or the nucleotide sequence of SEQ ID NO:41.

The present invention also provides a fusion protein comprising (a) afirst amino acid sequence comprising amino acid 42 to amino acid 60 ofSEQ ID NO:2, and (b) a second amino acid sequence derived from thesequence of a protein other than P-selectin ligand. Preferably, thefirst amino acid sequence comprises amino acid 42 to amino acid 402 ofSEQ ID NO:2, amino acid sequence comprises amino acid 42 to amino acid310 of SEQ ID NO:2, amino acid 42 to amino acid 88 of SEQ ID NO:2, aminoacid 42 to amino acid 118 of SEQ ID NO:2, or amino acid 42 to amino acid189 of SEQ ID NO:2.

In certain particularly preferred embodiments, the fusion proteincomprises the amino acid sequence of SEQ ID NO:36 from amino acid 42 toamino acid 313, the amino acid sequence of SEQ ID NO:36, the amino acidsequence of SEQ ID NO:38 from amino acid 42 to amino acid 269, the aminoacid sequence of SEQ ID NO:38, the amino acid sequence of SEQ ID NO:40from amino acid 42 to amino acid 437, the amino acid sequence of SEQ IDNO:40, the amino acid sequence of SEQ ID NO:42 from amino acid 42 toamino acid 264, or the amino acid sequence of SEQ ID NO:42.

In other preferred embodiments, the second amino acid sequence is linkedto the C-terminus or the N-terminus of the first amino acid sequence,with or without being linked by a linking sequence.

In yet other embodiments, the second amino acid sequence is derived froma protein selected from the group consisting of an antibody, a cytokine,a growth factor, a differentiation factor, a hormone, an enzyme, areceptor or fragment thereof and a ligand. Preferably, the second aminoacid sequence is derived from the sequence of an antibody, from the Fcportion of an antibody, or is a mutation of a sequence derived from anantibody.

In yet further embodiments, the present invention provides for acomposition comprising (a) a first peptide comprising amino acid 42 toamino acid 60 of SEQ ID NO:2, and (b) a second peptide derived from thesequence of a protein other than P-selectin ligand, wherein the firstpeptide and the second peptide are chemically linked by a moiety otherthan a peptide bond. Any P-selectin ligand protein of the invention canbe used in such a composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph comparing the binding of P-selectin ligand proteinsexpressed with and without core2.

FIG. 2 is an autoradiograph of immunoprecipitations of P-selectin ligandprotein expressed with and without core2.

FIG. 3 depicts the results of flow cytometry analysis of the binding ofP-selectin ligand protein (expressed with and without core2) toP-selectin/IgG chimera (LEC-γ1) and anti-P-selectin ligand proteinmonoclonal antibody (MAb 275).

FIG. 4 is an autoradiograph of proteins, including P-selectin ligandprotein, which bound to P- and E-selectin/IgG chimeras.

FIG. 5 is a schematic representation of structural features of the fulllength P-selectin ligand protein of SEQ ID NO: 2.

FIG. 6 is a schematic representation of several P-selectin ligandprotein fragments constructed for the purpose of examining the role ofN-linked glycosylation sites in binding of the P-selectin ligandproteins to selecting.

FIG. 7 depicts the results of experiments to determine the role ofN-linked glycosylation sites in binding of the P-selectin ligandproteins to selectins.

FIG. 8 is a schematic representation of several P-selectin ligandprotein fragments constructed for the purpose of examining the role ofsulfated tyrosine residues in binding of the P-selectin ligand proteinsto selecting.

FIGS. 9-11 depicts the results of experiments to determine the role ofsulfated tyrosine residues in binding of the P-selectin ligand proteinsto selecting.

FIG. 12 is a schematic representation of several P-selectin ligandprotein fragments constructed for the purpose of examining the effectsof various deletions on the binding of the P-selectin ligand proteins toselecting.

FIG. 13 is a schematic depiction of the quantitative plate binding assayof Example 4(c).

FIGS. 14-17 depict the results of experiments comparing the binding ofvarious deleted and altered P-selectin ligand proteins to selectins.

FIG. 18 is a schematic representation of several P-selectin ligandprotein fragments constructed for the purpose of examining the effectsof alteration of tyrosine residues in the anionic region of theP-selectin ligand proteins on selectin binding.

FIGS. 19-21 depict the results of experiments comparing the binding ofvarious deleted and altered P-selectin ligand proteins to selecting.

FIG. 22 depicts a proposed model for binding of P-selectin ligandproteins to P- and E-selectin.

FIGS. 23 and 24 depict the results of experiments comparing the bindingof various deleted and altered P-selectin ligand proteins to selecting.

Some of the foregoing figures employ a convention for numbering aminoacids within the depicted constructs which is different that the residuenumbering employed in SEQ ID NO:2. In the figures, residues are numberedusing the first amino acid of soluble mature P-selectin ligand as astarting point. Hence, the residue numbers used in the figures are 41less than those of SEQ ID NO:2. For example, residue 19 in the figurescorresponds to residue 60 in SEQ ID NO:2.

FIG. 25 is an analysis of the expression products of CHO cells, alreadyexpressing ¾ fucosyltransferase and Core2 transferase, which weretransfected with psPSL.T7, ΔTM, 1316 or psPSL.QC and amplified usingmethotrexate. Conditioned media was either analyzed directly or firstprecipitated with LEC-γ1 and then analyzed by SDS-PAGE undernon-reducing and reducing conditions.

FIG. 26. SDS-PAGE separation of myeloid cell membrane proteins affinitycaptured by P- and E-selectin. Membrane lysates were prepared from U937cells metabolically labeled with ³H-glucosamine and subjected toaffinity precipitation with immobilized P- and E-selectin and controlhuman IgG₁. Eluted proteins were treated with (“reduced”) or without(“non-reduced”) DTT prior to gel electrophoresis. Lanes: 1, affinitycapture by human IgG₁; 2, affinity capture by P-selectin; 3, affinitycapture by E-selectin.

FIG. 27. Sequential affinity capture experiments. ³H-labeled U937 lysatespecies were affinity captured by P- or E-selectin, eluted, and thensubjected to immunoprecipitation with anti-PSGL-1 antiserum Rb3443.Lanes: 1 and 2, control immunoprecipitations of fresh myeloid cellmembrane lysates using pre-immune rabbit serum (lane 1) and Rb3443 (lane2); 3-5, immunoprecipitation with Rb3443 of myeloid cell membranelysates previously affinity captured and eluted from P-selectin (lane3), E-selectin (lane 4), and human IgG₁ (lane 5).

FIG. 28. Comparison of CD43 and PSGL-1 content of myeloid cell membraneextracts. Labeled U937 cell extracts were immunoprecipitated withanti-PSGL-1 rabbit polyclonal antibody Rb3443 or an anti-CD43 mouse MAband then subjected to SDS-PAGE/autoradiography. Lanes: 1,immunoprecipitation with control pre-immune rabbit serum; 2,immunoprecipitation with Rb3443; 3, immunoprecipitation with controlisotype-matched mouse antibody; 4, immunoprecipitation with anti-CD43antibody.

FIG. 29. COS transfection experiments. COS M6 cells transfected withplasmids encoding PSGL-1 or CD43 as well as Fuc-TIII or Fuc-TVII weremetabolically labeled with ³⁵S-methionine, and membranes were preparedfor affinity capture experiments as described in Materials and Methods.The cDNAs employed in the transfections are indicated above the lanes.Precipitations were performed using (A) E-selectin, (B) P-selectin, and(C) anti-PSGL-1 antiserum Rb3443 and anti-CD43 MAb.

FIG. 30 summarizes the results of screening of various P-selecint ligandproteins for inhibition of P- and E-selectin binding (see Example 13).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have for the first time identified and isolated anovel DNA which encodes a protein which acts as a ligand for P-selectinon human endothelial cells and platelets. The sequence of the DNA is setforth in SEQ ID NO:1. The complete amino acid sequence of the P-selectinligand protein (i.e., the mature peptide plus the leader sequence) ischaracterized by the amino acid sequence set forth in SEQ ID NO:2 fromamino acid 1 to amino acid 402. Hydrophobicity analysis and comparisonwith known cleavage patterns predict a signal sequence of 20 to 22 aminoacids, i.e., amino acids 1 to 20 or amino acids 1 to 22 of SEQ ID NO:2.The P-selectin ligand protein contains a PACE (paired basic amino acidconverting enzyme) cleavage site (-Arg-Asp-Arg-Arg-) at amino acids 3841of SEQ ID NO:2. The mature P-selectin ligand protein of the presentinvention is characterized by the amino acid sequence set forth in SEQID NO:2 from amino acid 42 to amino acid 402. A soluble form of theP-selectin ligand protein is characterized by containing amino acids 21to 310 of SEQ ID NO:2. Another soluble form of the mature P-selectinligand protein is characterized by the amino acid sequence set forth inSEQ ID NO:2 from amino acid 42 to amino acid 310. The soluble form ofthe P-selectin ligand protein is further characterized by being solublein aqueous solution at room temperature. Of course, the correspondingDNA sequences as set forth in SEQ ID NO:1 encoding these proteins arealso included in the subject invention.

The P-selectin ligand of the invention is a glycoprotein which maycontain one or more of the following terminal carbohydrates:

where R=the remainder of the carbohydrate chain, which is covalentlyattached either directly to the P-selectin ligand protein or to a lipidmoiety which is covalently attached to the P-selectin ligand protein.The P-selectin ligand glycoprotein of the invention may additionally besulfated or otherwise post-translationally modified. As expressed in COSand CHO cells, full length P-selectin ligand protein (amino acids 1 to402 of SEQ ID NO:2 or amino acids 42 to 402 of SEQ ID NO:2) is ahomodimeric protein having an apparent molecular of 220 kD as shown bynon-reducing SDS-polyacrylamide gel electrophoresis.

The structure of the full-length P-selectin ligand protein isschematically represented in FIG. 5. Three regions of the P-selectinligand protein of SEQ ID NO:2 are: an extracellular domain (from aboutamino acid 21 to 310 of SEQ ID NO:2), a transmembrane domain (from aboutamino acid 311 to 332 of SEQ ID NO:2), and an intracellular, cytoplasmicdomain (from about amino acid 333 to 402 of SEQ ID NO:2). Theextracellular domain contains three consensus tripeptide sites(Asn-X-Ser/Thr) of potential N-linked glycosylation beginning at Asnresidues 65, 111, and 292. The extracellular domain further containsthree potential sites of tyrosine sulfation at residues 46, 48, and 51.The region comprised of residues 55-267 contains a high percentage ofproline, serine, and threonine including a subdomain of fifteendecameric repeats of the ten amino acid consensus sequenceAla-Thr/Met-Glu-Ala-Gln-Thr-Thr-X-Pro/Leu-Ala/Thr, wherein X can beeither Pro, Ala, Gln, Glu, or Arg. Regions such as these arecharacteristic of highly O-glycosylated proteins.

COS or CHO cells co-transfected with a gene encoding the P-selectinligand protein and a gene encoding fucosyltransferase (hereinafter FT),preferably an (α1,3/α1,4) fucosyltransferase (“3/4FT”), are capable ofbinding to CHO cells expressing P-selectin on their surface, but are notcapable of binding to CHO cells which do not express P-selectin on theirsurface. In order to bind to P-selectin, either in purified form orexpressed on the surface of CHO cells, the gene encoding the P-selectinligand protein must be co-transfected with the gene encoding an FT,since transfection of either gene in the absence of the other eitherabolishes or substantially reduces the P-selectin binding activity. Thebinding of the P-selectin ligand protein of the invention to P-selectincan be inhibited by EDTA or by a neutralizing monoclonal antibodyspecific for P-selectin. The binding of the P-selectin ligand protein ofthe invention to P-selectin is not inhibited by a non-neutralizingmonoclonal antibody specific for P-selectin or by an isotype control.These results characterize the binding specificity of the P-selectinligand protein of the invention.

For the purposes of the present invention, a protein is defined ashaving “P-selectin ligand protein activity”, i.e., variably referred toherein as a “P-selectin ligand protein”, or as a “P-selectin ligandglycoprotein” or simply as a “P-selectin ligand”, when it binds in acalcium-dependent manner to P-selectin which is present on the surfaceof cells as in the CHO-P-selectin binding assay of Example 4, or toP-selectin which is affixed to another surface, for example, as thechimeric P-selectin-IgGγ1 protein of Example 4 is affixed to Petridishes.

The glycosylation state of the P-selectin ligand protein of theinvention was studied using a chimeric, soluble form of the P-selectinligand protein, described in detail in Example 5(C) and designatedsPSL.T7. The sPSL.T7 protein produced from COS cells co-transfected with3/4FT is extensively modified by post-translational glycosylation, asdescribed in detail in Example 6(C). Thus, it is believed that both N-and O-linked oligosaccharide chains, at least some of which aresialated, are present on the P-selectin ligand protein of the invention.

The P-selectin ligand protein of the invention may also bind toE-selectin and L-selectin. Conditioned medium from COS cells which havebeen co-transfected with the DNA encoding sPSL.T7 or P-selectinligand-Ig fusions and with the DNA encoding 314FT, when coated on wellsof plastic microtiter plates, causes CHO cells which express E-selectinto bind to the plates; however CHO cells which do not express E-selectindo not bind to such plates. The binding of CHO cells which expressE-selectin to microtiter plates coated with conditioned medium from COScells which have been co-transfected with the DNA encoding sPSL.T7 andwith the DNA encoding 3/4FT is abolished in the presence of EDTA or of aneutralizing antibody specific for E-selectin. Conditioned medium fromCOS cells transfected only with the sPSL.T7 DNA does not cause bindingof CHO cells which express E-selectin when coated on wells of microtiterplates. For these reasons, the P-selectin ligand protein of theinvention is believed to be useful as an inhibitor ofE-selectin-mediated intercellular adhesion in addition toP-selectin-mediated intercellular adhesion.

Antibodies raised against COS-produced soluble P-selectin ligand proteinare immunoreactive with the major HL-60 glycoprotein that specificallybinds P-selectin as determined by affinity capture using an immobilizedFc chimera of P-selectin. U937 cells bear a similar immunoreactiveglycoprotein ligand. Thus, a single glycoprotein species is observedupon EDTA elution of immobilized P-selectin previously incubated withdetergent extracts of ³H-glucosamine labeled U937 cells. This majorspecies exhibits an apparent molecular weight by SDS-PAGE of 220 kDunder non-reducing conditions and 100 kD under reducing conditions. Aswith the comparable species isolated from HL-60 cells, this U937 ligandis immunoreactive with a polyclonal antibody raised against COSrecombinant P-selectin ligand protein. In addition, affinity capture ofE-selectin ligands from U937 cell and cell membrane preparations, usingan immobilized Fc chimera of E-selectin, yield a single major specieswith identical mass and electrophoretic behavior as the major U937P-selectin ligand. Thus, E- and P-selectin recognize the same majorglycoprotein ligand in U937 cells, a glycoprotein ligand immunoreactivewith an anti-P-selectin ligand protein antibody and possessing the sameapparent mass and electrophoretic behavior as full length, recombinantP-selectin ligand protein.

Fragments of the P-selectin ligand protein which are capable ofinteracting with P-selectin or which are capable of inhibitingP-selectin-mediated intercellular adhesion are also encompassed by thepresent invention. Such fragments comprise amino acids 21 to 54 of SEQID NO:2, a region of the P-selectin ligand protein having a lowfrequency of serine and threonine residues; amino acids 55 to 127 of SEQID NO:2, having a high frequency of proline, serine, and threonine inaddition to two consensus sequences for asparagine-linked glycosylation(Asn-X-Ser/Thr); another larger fragment, amino acids 128 to 267 of SEQID NO:2, having both a high frequency of proline, serine, and threonineand containing fifteen repeats of the following ten amino acid consensussequence:Ala-(Thr/Met)-Glu-Ala-Gln-Thr-Thr-(Pro/Arg/Gln/Ala/Glu)-(Leu/Pro)-(Ala/Thr)(smaller fragments within this large fragment may also retain thecapacity to interact with P-selectin or act as inhibitors ofP-selectin-mediated intercellular adhesion); the region containing aconsensus sequence for asparagine-linked glycosylation and comprisingamino acids 268 to 308 of SEQ ID NO:2; the hydrophobic region of theprotein represented by amino acids 309 to 333 of SEQ ID NO:2; and theamphophilic region of the P-selectin ligand protein from amino acids 334to 402 of SEQ ID NO:2. Additional fragments may comprise amino acid 43to amino acid 56 of SEQ ID NO:2 or amino acid 42 to amino acid 60 of SEQID NO:2, with one or more sulfated or phosphorylated (Domcheck et al.,Biochemistry 31:9865-9870 (1992)) tyrosines at amino acid 46, amino acid48, and/or amino acid 51. Fragments of the P-selectin ligand protein maybe in linear form or they may be cyclized using known methods, forexample, as described in H. U. Saragovi, et al., BioTechnology 10,773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114,9245-9253 (1992), both of which are incorporated herein by reference.For the purposes of the present invention, all references to “P-selectinligand protein” herein include fragments capable of binding toP-selectin.

Such fragments may be fused to carrier molecules such asimmunoglobulins, to increase the valency of P-selectin ligand bindingsites. For example, soluble forms of the P-selectin ligand protein suchas the fragments from amino acid 42 to amino acid 295 or from amino acid42 to amino acid 88 of SEQ ID NO:2 may be fused through “linker”sequences to the Fc portion of an immunoglobulin (native sequence ormutated sequences for conferring desirable qualities (such as longerhalf-life or reduced immunogenicity) to the resulting chimera). For abivalent form of the P-selectin ligand protein, such a fusion could beto the Fc portion of an IgG molecule as in Example 5(D) and in SEQ IDNO:6. Other immunoglobulin isotypes may also be used to generate suchfusions. For example, a P-selectin ligand protein—IgM fusion wouldgenerate a decavalent form of the P-selectin ligand protein of theinvention.

Fusions of any of the P-selectin ligand proteins of the presentinvention to amino acid sequences derived from other proteins may alsobe constructed. Preferred P-selectin ligand proteins for such purposeinclude the fragments from amino acid 42 to amino acid 295 or from aminoacid 42 to amino acid 88 of SEQ ID NO:2. Desirable fusion proteins mayincorporate amino acid sequence from proteins having a biologicalactivity different from that of P-selectin ligand, such as, for example,cytokines, growth and differentiation factors (such as bonemorphogenetic proteins (e.g., BMPs), hormones, enzymes, receptorcomponents or fragments and other ligands. Also, P-selectin ligandprotein can be chemically coupled to other proteins or pharmaceuticalagents. In such usage, the P-selectin ligand protein, by virtue of theability to interact with selectin molecules, alters the pharmacokineticsand/or biodistribution of the fused or coupled agent thereby enhancingits therapeutic efficacy. For example, fusion of a P-selectin ligandprotein sequence to a cytokine sequence can direct the cytokine'sactivity to an area of inflammation. In such instance, the P-selectinligand protein portion of the fusion protein will bind to selectinsexpressed at the site of inflammation. This binding will cause thecytokine portion of the fusion protein to become localized and availableto bind its cognate receptor or any proximal cell surface. Other ligandscould similarly be used in such fusions proteins to attract cellsexpressing their corresponding receptors to a site of P-selectinexpression. Preferred examples of such fusions are described in Example15.

In any fusion protein incorporating a P-selectin ligand protein, theamino acid sequence derived from a protein or proteins other thanP-selectin ligand can be linked to either the C-terminus or N-terminusof the P-selectin ligand-derived sequence. The linkage may be direct(i.e., without an intervening linking sequence not derived from eitherprotein) or through a linking sequence.

Methods of treating a mammalian subject using such fusion proteins arealso contemplated by the present invention. In such instances, thefusion protein is used to treat a condition which is affected by theprotein to which the P-selectin ligand protein is fused. For example, afusion of a P-selectin ligand protein to IL-11 could be used to localizethe activity of IL-11 to bone marrow endothelial cells which expressselectins on their surface. Once localized, the IL-11 portion of thefusion protein will stimulate megakaryocyte progenitors. Similarly, afusion of a P-selectin ligand protein to a BMP could be used tostimulate bone or cartilage formation in an area of injury. Injuredtissues express P-selectin, which will bind the fusion protein. Oncelocalized, the BMP portion of the fusion protein will stimulate bone orcartilage production in the area of injury.

As detailed in the Examples below, the P-selectin ligand protein of theinvention was initially obtained using an expression cloning approach(Clark et al., U.S. Pat. No. 4,675,285). A cDNA library was constructedfrom the human promyelocytic cell line HL-60 (S. J. Collins, et al.,Nature 270, 347-349 (1977), ATCC No. CCL 240). This library wascotransfected into COS cells with a DNA encoding a 3/4FT, and thecotransfectants were screened for binding to a chimeric moleculeconsisting of the extracellular portion of P-selectin and the Fc portionof a human IgGγ1 monoclonal antibody. Cotransfectants which bound to thechimeric P-selectin were enriched for cDNAs encoding the P-selectinligand protein. This screening process was repeated several times toenrich the plasmid population further for cDNAs encoding the P-selectinligand protein. In a second cloning stage, the enriched plasmidpopulation was again cotransfected into COS cells with the 3/4FT geneand screened for binding to a fluorescently labeled CHO cell line whichexpressed P-selectin on the cell surface. A single cDNA clone wasobtained from this approach and was designated pMT21:PL85. ThepMT21:PL85 plasmid was deposited with the American Type CultureCollection on Oct. 16, 1992 and given the accession number ATCC 69096.

One novel DNA of the present invention is set forth in SEQ ID NO:1. TheDNA of the present invention may encode a variety of forms of theP-selectin ligand protein. For example, in one embodiment, the DNA ofthe invention encodes the entire P-selectin ligand protein having theamino acid sequence set forth in SEQ ID NO:2 from amino acid 1 to aminoacid 402. In another embodiment, the DNA of the invention encodes a formof the P-selectin ligand protein which lacks the signal sequence andwhich is characterized by the amino acid sequence set forth in SEQ IDNO:2 from amino acid 21 to amino acid 402. In yet another embodiment,the DNA of the invention encodes the mature P-selectin ligand proteincharacterized by the amino acid sequence set forth in SEQ ID NO:2 fromamino acid 42 to amino acid 402. Another embodiment of the DNA of theinvention encodes a soluble form of the P-selectin ligand proteincharacterized by the amino acid sequence set forth in SEQ ID NO:2 fromamino acid 1 to amino acid 310. The DNA of the invention is alsoembodied in a DNA encoding a soluble form of the mature P-selectinligand protein, said protein being characterized by the amino acidsequence set forth in SEQ ID NO:2 from amino acid 42 to amino acid 310.The DNA of the invention is further embodied in a DNA sequence encodinga soluble form of the P-selectin ligand protein which lacks the signalsequence, said protein being characterized by the amino acid sequenceset forth in SEQ ID NO:2 from amino acid 21 to amino acid 310. The DNAof the present invention is free from association with other human DNAsand is thus characterized as an isolated DNA. As detailed above, DNAswhich encode P-selectin ligand fragments which interact with P-selectinare also included in the present invention.

The expression of P-selectin ligand protein mRNA transcripts has beenobserved in a variety of human cell lines (HL-60, THP-1, U937) and inhuman monocytes and polymorphonuclear leukocytes by Northern analysisusing a P-selectin ligand protein cDNA probe. In all of these celllines, a major transcript of 2.5 kb was observed. A minor species ofapproximately 4 kb was observed in the HL60 and U937 cell lines and inpolymorphonuclear leukocytes. In contrast, no P-selectin ligand mRNAexpression was detected in the human hepatoblastoma cell line HepG2.

The P-selectin ligand protein of the invention is encoded by a singlecopy gene and is not part of a multi-gene family, as determined bySouthern blot analysis. The genomic form of the P-selectin ligandprotein of the invention contains a large intron of approximately 9 kblocated at nucleotide 54 in the 5′ untranslated region. Inpolymorphonuclear leukocytes and monocytes, the P-selectin ligandprotein of the invention is encoded by the DNA sequence set forth in SEQID NO:3. In this embodiment, the P-selectin ligand protein containssixteen repeat regions. The isolated DNA of the invention iscorrespondingly also embodied in the DNA sequence set forth in SEQ IDNO:3 and is contained on plasmid pPL85R16 which was deposited with theAmerican Type Culture Collection on Oct. 22, 1993 and given theAccession Number ATCC 75577.

The invention also encompasses allelic variations of the isolated DNA asset forth in SEQ ID NO:1 or of the isolated DNA as set forth in SEQ IDNO:3, that is, naturally-occurring alternative forms of the isolated DNAof SEQ ID NO:1 or SEQ ID NO:3 which also encode proteins havingP-selectin ligand activity. Also included in the invention are isolatedDNAs which hybridize to the DNA set forth in SEQ ID NO:1 or to the DNAset forth in SEQ ID NO:3 under stringent (e.g. 4×SSC at 65° C. or 50%formamide and 4×SSC at 42° C.), or relaxed (4×SSC at 50° C. or 30-40%formamide at 42° C.) conditions, and which have P-selectin ligandprotein activity. Isolated DNA sequences which encode the P-selectinligand protein but which differ from the DNA set forth in SEQ ID NO:1 orfrom the DNA set forth in SEQ ID NO:3 by virtue of the degeneracy of thegenetic code and which have P-selectin ligand protein activity are alsoencompassed by the present invention. Variations in the DNA as set forthin SEQ ID NO:1 or in the DNA as set forth in SEQ ID NO:3 which arecaused by point mutations or by induced modifications which enhance theP-selectin ligand activity, half-life or production level are alsoincluded in the invention. For the purposes of the present invention allreferences herein to the “DNA of SEQ ID NO:1” include, in addition toDNAs comprising the specific DNA sequence set forth in SEQ ID NO:1, DNAsencoding the mature P-selectin ligand protein of SEQ ID NO:2; DNAsencoding fragments of the P-selectin ligand protein of SEQ ID NO:2 whichare capable of binding to P-selectin; DNAs encoding soluble forms of theP-selectin ligand protein of SEQ ID NO:2; allelic variations of the DNAsequence of SEQ ID NO:1; DNAs which hybridize to the DNA sequence of SEQID NO:1 and which encode proteins having P-selectin ligand proteinactivity; DNAs which differ from the DNA of SEQ ID NO:1 by virtue ofdegeneracy of the genetic code; and the variations of the DNA sequenceof SEQ ID NO:1 set forth above. Similarly, all references to the “DNA ofSEQ ID NO:3” include in addition to the specific sequence set forth inSEQ ID NO:3, DNAs encoding the mature P-selectin ligand protein of SEQID NO:4; DNAs encoding fragments of the P-selectin ligand protein of SEQID NO:4 which are capable of binding to P-selectin; DNAs encodingsoluble forms of the P-selectin ligand protein of SEQ ID NO:4; allelicvariations of the DNA of SEQ ID NO:3; DNAs which hybridize to the DNAsequence of SEQ ID NO:3 and which encode proteins having P-selectinligand protein activity; DNAs which differ from the DNA of SEQ ID NO:3by virtue of degeneracy of the genetic code; and the variations of theDNA of SEQ ID NO:3 set forth above.

A DNA encoding a soluble form of the P-selectin ligand protein may beprepared by expression of a modified DNA in which the regions encodingthe transmembrane and cytoplasmic domains of the P-selectin ligandprotein are deleted and/or a stop codon is introduced 3′ to the codonfor the amino acid at the carboxy terminus of the extracellular domain.For example, hydrophobicity analysis predicts that the P-selectin ligandprotein set forth in SEQ ID NO:2 has a transmembrane domain comprised ofamino acids 311 to 332 of SEQ ID NO:2 and a cytoplasmic domain comprisedof amino acids 333 to 402 of SEQ ID NO:2. A modified DNA as describedabove may be made by standard molecular biology techniques, includingsite-directed mutagenesis methods which are known in the art or by thepolymerase chain reaction using appropriate oligonucleotide primers.Methods for producing several DNAs encoding various soluble P-selectinligand proteins are set forth in Example 5.

A DNA encoding other fragments and altered forms of P-selectin ligandprotein may be prepared by expression of modified DNAs in which portionsof the full-length sequence have been deleted or altered. Substantialdeletions of the P-selectin ligand protein sequence can be made whileretaining P-selectin ligand protein activity. For example, P-selectinligand proteins comprising the sequence from amino acid 42 to amino acid189 of SEQ ID NO: 2, the sequence from amino acid 42 to amino acid 118of SEQ ID NO: 2, or the sequence from amino acid 42 to amino acid 89 ofSEQ ID NO: 2 each retain the P-selectin protein binding activity and theability to bind to E-selectin. P-selectin ligand proteins in which oneor more N-linked glycosylation sites (such as those at amino acids 65,111 and 292 of SEQ ID NO: 2) have been changed to other amino acids ordeleted also retain P-selectin protein binding activity and the abilityto bind E-selectin. P-selectin ligand proteins comprising from aminoacid 42 to amino acid 60 of SEQ ID NO:2 (which includes a highly anionicregion of the protein from amino acid 45 to amino acid 58 of SEQ IDNO:2) also retain P-selectin ligand protein activity; however,P-selectin ligand proteins limited to such sequence do not bind toE-selectin. Preferably, a P-selectin ligand protein retains at least one(more preferably at least two and most preferably all three) of thetyrosine residues found at amino acids 46, 48 and 51 of SEQ ID NO: 2,sulfation of which may contribute to P-selectin ligand protein activity.Construction of DNAs encoding these and other active fragments oraltered forms of P-selectin ligand protein may be accomplished inaccordance with methods known to those skilled in the art.

The isolated DNA of the invention may be operably linked to anexpression control sequence such as the pMT2 or pED expression vectorsdisclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), inorder to produce the P-selectin ligand recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified in R.Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein“operably linked” means enzymatically or chemically ligated to form acovalent bond between the isolated DNA of the invention and theexpression control sequence, in such a way that the P-selectin ligandprotein is expressed by a host cell which has been transformed(transfected) with the ligated DNA/expression control sequence.

Several endoproteolytic enzymes are known which cleave precursorpeptides at the carboxyl side of paired amino acid sequences (e.g.,-Lys-Arg- and -Arg-Arg-) to yield mature proteins. Such enzymes aregenerally known as paired basic amino acid converting enzymes or PACE,and their use in recombinant production of mature peptides isextensively disclosed in WO 92/09698 and U.S. application Ser. No.07/885,972, both of which are incorporated herein by reference. The PACEfamily of enzymes are known to increase the efficiency of proteolyticprocessing of precursor polypeptides in recombinant host cells. Asmentioned above, the P-selectin ligand protein of the invention containssuch a PACE cleavage site.

The soluble mature P-selectin ligand protein of the present inventionmay be made by a host cell which contains a DNA sequence encoding anysoluble P-selectin ligand protein as described herein and a DNA sequenceencoding PACE as described in WO 92/09698 and U.S. application Ser. No.07/885,972, incorporated herein by reference, or using the DNA sequenceof SEQ ID NO:5. Such a host cell may contain the DNAs as the result ofco-transformation or sequential transformation of separate expressionvectors containing the soluble P-selectin ligand protein DNA and thePACE DNA, respectively. A third DNA which encodes a 3/4FT may also beco-transformed with the DNAs encoding the P-selectin ligand protein andPACE. Alternatively, the host cell may contain the DNAs as the result oftransformation of a single expression vector containing both solubleP-selectin ligand protein DNA and PACE DNA. Construction of suchexpression vectors is within the level of ordinary skill in molecularbiology. Methods for co-transformation and transformation are alsoknown.

Many DNA sequences encoding PACE are known. For example, a DNA encodingone form of PACE, known as furin, is disclosed in A. M. W. van denOuweland et al., Nucl. Acids Res. 18, 664 (1990), incorporated herein byreference. A cDNA encoding a soluble form of PACE, known as PACESOL, isset forth in SEQ ID NO:5. DNAs encoding other forms of PACE also exist,and any such PACE-encoding DNA may be used to produce the soluble matureP-selectin ligand protein of the invention, so long as the PACE iscapable of cleaving the P-selectin ligand protein at amino acids 38-41.Preferably, a DNA encoding a soluble form of PACE is used to produce thesoluble mature P-selectin ligand protein of the present invention.

The DNAs encoding a soluble form of the P-selectin ligand protein andPACE, separately or together, may be operably linked to an expressioncontrol sequence such as those contained in the pMT2 or pED expressionvectors discussed above, in order to produce the PACE-cleaved solubleP-selectin ligand recombinantly. Additional suitable expression controlsequences are known in the art. Examples 3(C) and 3(D) below set forthmethods for producing the soluble mature P-selectin ligand protein ofthe invention.

A number of types of cells may act as suitable host cells for expressionof the P-selectin ligand protein. Suitable host cells are capable ofattaching carbohydrate side chains characteristic of functionalP-selectin ligand protein. Such capability may arise by virtue of thepresence of a suitable glycosylating enzyme within the host cell,whether naturally occurring, induced by chemical mutagenesis, or throughtransfection of the host cell with a suitable expression plasmidcontaining a DNA sequence encoding the glycosylating enzyme. Host cellsinclude, for example, monkey COS cells, Chinese Hamster Ovary (CHO)cells, human kidney 293 cells, human epidermal A431 cells, human Colo205cells, 3T3 cells, CV-1 cells, other transformed primate cell lines,normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60,U937, or HaK cells.

The P-selectin ligand protein may also be produced by operably linkingthe isolated DNA of the invention and one or more DNAs encoding suitableglycosylating enzymes to suitable control sequences in one or moreinsect expression vectors, and employing an insect expression system.Materials and methods for baculovirus/insect cell expression systems arecommercially available in kit form from, e.g., Invitrogen, San Diego,Calif., U.S.A. (the MaxBac® kit), and such methods are well known in theart, as described in Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987), incorporated herein by reference.Soluble forms of the P-selectin ligand protein may also be produced ininsect cells using appropriate isolated DNAs as described above. A DNAencoding a form of PACE may further be co-expressed in an insect hostcell to produce a PACE-cleaved form of the P-selectin ligand protein.

Alternatively, it may be possible to produce the P-selectin ligandprotein in lower eukaryotes such as yeast or in prokaryotes such asbacteria. Potentially suitable yeast strains include Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida,or any yeast strain capable of expressing heterologous proteins.Potentially suitable bacterial strains include Escherichia coli,Bacillus subtilis, Salmonella typhimurium, or any bacterial straincapable of expressing heterologous proteins. If the P-selectin ligandprotein is made in yeast or bacteria, it is necessary to attach theappropriate carbohydrates to the appropriate sites on the protein moietycovalently, in order to obtain the glycosylated P-selectin ligandprotein. Such covalent attachments may be accomplished using knownchemical or enzymatic methods.

The P-selectin ligand protein of the invention may also be expressed asa product of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a DNA sequence encoding the P-selectinligand protein.

The P-selectin binding activity of a P-selectin protein may be enhancedby co-transformation of a host cell with a GlcNAc transferase,preferably UDP-GlcNAc:Gal β1-3GalNAc-R(GlcNAc to GalNAc)β1-6 GlcNActransferase (EC 2.4.1.102), also known as “core2 transferase.”

O-linked glycans present on P-selectin ligand protein have been shown tobe important for binding to P-selectin (D. Sako et al., Cell 75,1179-1186 (1993)). It has been reported that sialyl Le^(x) on O-linkedglycans of myeloid cells are presented on complex, branched structures(Maemura, K. and Fukuda, M., J. Biol. Chem. 267, 24379-24386 (1992)).The enzyme responsible for generating such oligosaccharide structures is“core2”. The core2 enzyme activity is found at very low levels in COScells and at trace levels in CHO cells. Host cells co-transformed withDNAs encoding a P-selectin ligand protein, an (α1,3/α1,4)fucosyltransferase and core2 can produce P-selectin ligand proteinexhibiting 20-30 fold enhanced binding to P-selectin.

In certain preferred embodiments, P-selectin ligand protein is producedby co-transfecting a host cell with DNAs encoding soluble P-selectinligand protein, 3/4FT, core2 and PACE.

The P-selectin ligand protein of the invention may be prepared byculturing transformed host cells under culture conditions necessary toexpress a P-selectin binding glycoprotein. The resulting expressedglycoprotein may then be purified from culture medium or cell extracts.Soluble forms of the P-selectin ligand protein of the invention can bepurified by affinity chromatography over Lentil lectin-Sepharose® andsubsequent elution with 0.5M α-methyl-mannoside. The eluted solubleP-selectin ligand protein can then be further purified and concentratedby a 0-70% ammonium sulfate precipitation step. The protein is thenrecovered, resuspended, and further purified by size exclusionchromatography over a TSK G4000SW_(XL). Alternatively, full length formsof the P-selectin ligand protein of the invention can be purified bypreparing a total membrane fraction from the expressing cell andextracting the membranes with a non-ionic detergent such as TritonX-100. The detergent extract can then be passed over an affinity columncomprised of immobilized P-selectin, and the P-selectin ligand proteincan be eluted from the column with 10 mM EDTA in a buffer containing0.1% detergent. The material eluted from the affinity column can then bedialyzed to remove EDTA and further purified over a Lentillectin-Sepharose® affinity column again eluting with 0.5Mα-methyl-mannoside.

Alternatively, the P-selectin ligand protein of the invention isconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. Sulfopropylgroups are preferred (e.g., S-Sepharose® columns). The purification ofthe P-selectin ligand protein from culture supernatant may also includeone or more column steps over such affinity resins as concanavalinA-agarose, Heparin-Toyopearl® or Cibacrom blue 3GA Sepharose®; or byhydrophobic interaction chromatography using such resins as phenylether, butyl ether, or propyl ether; or by immunoaffinitychromatography.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the P-selectin ligand protein. Some or all ofthe foregoing purification steps, in various combinations, can also beemployed to provide a substantially homogeneous isolated recombinantprotein. The P-selectin ligand protein thus purified is substantiallyfree of other mammalian proteins and is defined in accordance with thepresent invention as “isolated P-selectin ligand protein”.

Isolated P-selectin ligand protein may be useful in treating conditionscharacterized by P-, E- or L-selectin mediated intercellular adhesion.Such conditions include, without limitation, myocardial infarction,bacterial or viral infection, metastatic conditions, inflammatorydisorders such as arthritis, gout, uveitis, acute respiratory distresssyndrome, asthma, emphysema, delayed type hypersensitivity reaction,systemic lupus erythematosus, thermal injury such as burns or frostbite,autoimmune thyroiditis, experimental allergic encephalomyelitis,multiple sclerosis, multiple organ injury syndrome secondary to trauma,diabetes, Reynaud's syndrome, neutrophilic dermatosis (Sweet'ssyndrome), inflammatory bowel disease, Grave's disease,glomerulonephritis, gingivitis, periodontitis, hemolytic uremicsyndrome, ulcerative colitis, Crohn's disease, necrotizingenterocolitis, granulocyte transfusion associated syndrome,cytokine-induced toxicity, and the like Isolated P-selectin ligandprotein may also be useful in organ transplantation, both to prepareorgans for transplantation and to quell organ transplant rejection.Accordingly, P-selectin ligand protein may be administered to a livingor non-living organ donor, prior to organ removal. In addition,P-selectin ligand protein may be administered “ex-vivo” to the donororgan concomitantly with organ preservation solution, prior to, and/orsubsequent to surgical anastomosis with the recipient. IsolatedP-selectin ligand protein may be used to treat hemodialysis andleukophoresis patients. Additionally, isolated P-selectin ligand proteinmay be used as an antimetastatic agent. Isolated P-selectin ligandprotein may be used itself as an inhibitor of P-, E- orL-selectin-mediated intercellular adhesion or to design inhibitors ofP-, E- or L-selectin-mediated intercellular adhesion. The presentinvention encompasses both pharmaceutical compositions containingisolated P-selectin ligand protein and therapeutic methods of treatmentor use which employ isolated P-selectin ligand protein.

Isolated P-selectin ligand protein, purified from cells or recombinantlyproduced, may be used as a pharmaceutical composition when combined witha pharmaceutically acceptable carrier. Such a composition may contain,in addition to P-selectin ligand protein and carrier, diluents, fillers,salts, buffers, stabilizers, solubilizers, and other materials wellknown in the art. The term “pharmaceutically acceptable” means anon-toxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. Thepharmaceutical composition of the invention may also contain cytokines,lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,G-CSF, Meg-CSF, stem cell factor, and erythropoietin. The pharmaceuticalcomposition may contain thrombolytic or anti-thrombotic factors such asplasminogen activator and Factor VIII. The pharmaceutical compositionmay further contain other anti-inflammatory agents. Such additionalfactors and/or agents may be included in the pharmaceutical compositionto produce a synergistic effect with isolated P-selectin ligand protein,or to minimize side effects caused by the isolated P-selectin ligandprotein. Conversely, isolated P-selectin ligand protein may be includedin formulations of the particular cytokine, lymphokine, otherhematopoietic factor, thrombolytic or anti-thrombotic factor, oranti-inflammatory agent to minimize side effects of the cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent.

The pharmaceutical composition of the invention may be in the form of aliposome in which isolated P-selectin ligand protein is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers which inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S.Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No.4,737,323, all of which are incorporated herein by reference.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,healing of chronic conditions characterized by P-selectin- orE-selectin-mediated cellular adhesion or increase in rate of healing ofsuch conditions. When applied to an individual active ingredient,administered alone, the term refers to that ingredient alone. Whenapplied to a combination, the term refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of isolated P-selectin ligand proteinis administered to a mammal having a P-selectin-mediated disease state.Isolated P-selectin ligand protein may be administered in accordancewith the method of the invention either alone or in combination withother therapies such as treatments employing receptor antagonists,ligand antagonists, cytokines, lymphokines or other hematopoieticfactors. When co-administered with one or more cytokines, lymphokines orother hematopoietic factors, isolated P-selectin ligand protein may beadministered either simultaneously with the cytokine(s), lymphokine(s),other hematopoietic factor(s), thrombolytic or anti-thrombotic factors,or sequentially. If administered sequentially, the attending physicianwill decide on the appropriate sequence of administering isolatedP-selectin ligand protein in combination with cytokine(s),lymphokine(s), other hematopoietic factor(s), thrombolytic oranti-thrombotic factors.

Administration of isolated P-selectin ligand protein used in thepharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, or cutaneous, subcutaneous, or intravenousinjection. Intravenous administration to the patient is preferred.

When a therapeutically effective amount of isolated P-selectin ligandprotein is administered orally, isolated P-selectin ligand protein willbe in the form of a tablet, capsule, powder, solution or elixir. Whenadministered in tablet form, the pharmaceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%isolated P-selectin ligand protein, and preferably from about 25 to 90%isolated P-selectin ligand protein. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of isolated P-selectin ligand protein and preferably from about 1to 50% isolated P-selectin ligand protein.

When a therapeutically effective amount of isolated P-selectin ligandprotein is administered by intravenous, cutaneous or subcutaneousinjection, isolated P-selectin ligand protein will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to isolatedP-selectin ligand protein an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additive known to those of skill in the art.

The amount of isolated P-selectin ligand protein in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of isolated P-selectin ligand proteinwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of isolated P-selectin ligandprotein and observe the patient's response. Larger doses of isolatedP-selectin ligand protein may be administered until the optimaltherapeutic effect is obtained for the patient, and at that point thedosage is not increased further. It is contemplated that the variouspharmaceutical compositions used to practice the method of the presentinvention should contain about 0.1 μg to about 100 mg of isolatedP-selectin ligand protein per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the isolated P-selectin ligand proteinwill be in the range of 12 to 24 hours of continuous intravenousadministration. Ultimately the attending physician will decide on theappropriate duration of intravenous therapy using the pharmaceuticalcomposition of the present invention.

Isolated P-selectin ligand protein of the invention may also be used toimmunize animals to obtain polyclonal and monoclonal antibodies whichspecifically react with the P-selectin ligand protein and which mayinhibit P-selectin-mediated cellular adhesion. Such antibodies may beobtained using the entire P-selectin ligand protein as an immunogen, orby using fragments of P-selectin ligand protein such as the solublemature P-selectin ligand protein. Smaller fragments of the P-selectinligand protein may also be used to immunize animals, such as thefragments set forth below: amino acid 42 to amino acid 56 of SEQ ID NO:2and amino acid 127 to amino acid 138 of SEQ ID NO:2. An additionalpeptide immunogen comprises amino acid 238 to amino acid 248 of SEQ IDNO:2, with an alanine residue added to the amino terminus of thepeptide. Another peptide immunogen comprises amino acid 43 to amino acid56 of SEQ ID NO:2 having a sulfated tyrosine in any or all of positions46, 48 or 51. The peptide immunogens additionally may contain a cysteineresidue at the carboxyl terminus, and are conjugated to a hapten such askeyhole limpet hemocyanin (KLH). Additional peptide immunogens may begenerated by replacing tyrosine residues with sulfated tyrosineresidues. Methods for synthesizing such peptides are known in the art,for example, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154(1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987).

Monoclonal antibodies binding to P-selectin ligand glycoprotein or tocomplex carbohydrate moieties characteristic of the P-selectin ligandglycoprotein may be useful diagnostic agents for the immunodetection ofinflammatory diseases and some forms of cancer. Some cancerous cells,such as small cell lung carcinomas, may express detectable levels of theP-selectin ligand protein. This abnormal expression of the P-selectinligand protein by cancer cells may play a role in the metastasis ofthese cells.

Neutralizing monoclonal antibodies binding to P-selectin ligandglycoprotein or to complex carbohydrates characteristic of P-selectinligand glycoprotein may also be useful therapeutics for bothinflammatory diseases and also in the treatment of some forms of cancerwhere abnormal expression of P-selectin ligand protein is involved.These neutralizing monoclonal antibodies are capable of blocking theselectin mediated intercellular adherence function of the P-selectinligand protein. By blocking the binding of P-selectin ligand protein,the adherence of leukocytes to sites of inappropriate inflammation iseither abolished or markedly reduced. In the case of cancerous cells orleukemic cells, neutralizing monoclonal antibodies against P-selectinligand protein may be useful in detecting and preventing the metastaticspread of the cancerous cells which may be mediated by the P-selectinligand protein. In addition, the monoclonal antibodies bound to thesecells may target the cancerous cells for antibody-dependent cellmedicated cytoxicity (ADCC), thus helping to eliminate the cancerouscells. Human antibodies which react with the P-selectin ligand proteinmay be produced in transgenic animals which contain human immunoglobulinencoding genes in their germ lines. Example 7 below sets forthproduction of a rabbit polyclonal antibody specific P-selectin ligandprotein fragments.

P-selectin ligand protein of the invention may also be used to screenfor agents which are capable of binding to P-selectin ligand protein andthus may act as inhibitors of P-selectin- or E-selectin-mediatedintercellular adhesion. Binding assays using a desired binding protein,immobilized or not, are well known in the art and may be used for thispurpose using the P-selectin ligand protein of the invention.Appropriate screening assays may be cell-based, as in Examples 3 and 9below. Alternatively, purified protein based screening assays may beused to identify such agents. For example, P-selectin ligand protein maybe immobilized in purified form on a carrier and binding to purifiedP-selectin may be measured in the presence and in the absence ofpotential inhibiting agents. A suitable binding assay may alternativelyemploy purified P-selectin immobilized on a carrier, with a soluble formof P-selectin ligand protein of the invention.

Any P-selectin ligand protein may be used in the screening assaysdescribed above. For example, the full-length P-selectin ligand proteinset forth in SEQ ID NO:2 from amino acid 1 to amino acid 402 may be usedto screen for inhibitors; or the mature P-selectin ligand protein setforth in SEQ ID NO:2 from amino acid 42 to amino acid 402 may be used toscreen for inhibitors, or the soluble mature P-selectin ligand proteinset forth in SEQ ID NO:2 from amino acid 42 to amino acid 310 may beused to screen for inhibitors. Alternatively, the P-selectin ligandprotein of SEQ ID NO:4 from amino acid 1 to amino acid 412, or a matureform of the P-selectin ligand protein as set forth in SEQ ID NO:4 fromamino acid 42 to amino acid 412, or a soluble mature form of theP-selectin ligand protein set forth in SEQ ID NO:4 from amino acid 42 toamino acid 320 may be used to screen for inhibitors of intercellularadhesion in accordance with the present invention.

In such a screening assay, a first binding mixture is formed bycombining P-selectin or E-selectin and P-selectin ligand protein, andthe amount of binding in the first binding mixture (B_(o)) is measured.A second binding mixture is also formed by combining P-, E- orL-selectin, P-selectin ligand protein, and the compound or agent to bescreened, and the amount of binding in the second binding mixture (B) ismeasured. The amounts of binding in the first and second bindingmixtures are compared, for example, by performing a B/B_(o) calculation.A compound or agent is considered to be capable of inhibiting P-, E- orL-selectin mediated intercellular adhesion if a decrease in binding inthe second binding mixture as compared to the first binding mixture isobserved. The formulation and optimization of binding mixtures is withinthe level of skill in the art, such binding mixtures may also containbuffers and salts necessary to enhance or to optimize binding, andadditional control assays may be included in the screening assay of theinvention.

Compounds found to reduce by at least about 10%, preferably greater thanabout 50% or more of the binding activity of P-selectin ligand proteinto P-, E- or L-selectin may thus be identified and then secondarilyscreened in other selectin binding assays, including assays binding toL-selectin and in vivo assays. By these means compounds havinginhibitory activity for selectin-mediated intercellular adhesion whichmay be suitable as anti-inflammatory agents may be identified.

EXAMPLE 1 Cloning of the P-Selectin Ligand Protein Gene

A. Construction of the HL60 cDNA library

An HL60 cDNA library was constructed for expression cloning theP-selectin ligand. PolyA⁺ RNA was isolated from total RNA from the humanpromyelocytic cell line HL60 (S. J. Collins, et al., supra) using a FastTrack mRNA Isolation Kit (Invitrogen; San Diego, Calif.). Doublestranded cDNA was synthesized from the polyA⁺ RNA fraction and blunt-endligated with EcoRI adaptors (5′-AATTCCGTCGACTCTAGAG-3′, SEQ ID NO:7;5′-CTCTAGAGTCGACGG-3′, SEQ ID NO:8). The cDNA was ligated into theexpression vector pMT21 (R. Kaufman et al., J. Mol. Cell. Biol. 9,946-958 (1989) that had been incubated sequentially with EcoRIendonuclease and calf intestinal alkaline phosphatase and gel purified.The ligation product was electroporated in 2 μl aliquots into competentE. coli DH5α cells and grown in 1 ml of SOB medium (J. Sambrook et al.,Molecular Cloning: A Laboratory Manual, New York, Cold Spring HarborLaboratory Press, p 1.90 (1989)) which has been supplemented with 10 mMMgCl₂, 10 mM MgSO₄, and 2% glycerol for one hour at 37° C. In order todivide the library into smaller subsets, an aliquot from each ml ofbacterial suspension was plated onto agar plates in the presence ofampicillin, and the number of colonies per ml was calculated. Assumingthat each colony represented one cDNA clone, 600,000 clones weregenerated and divided into subsets of approximately 16,000 clones perpool. Each of the 38 pools were grown overnight in L-broth in thepresence of ampicillin and the plasmids were purified over a CsClgradient.

B. Screening for the P-Selectin Ligand Protein Gene

In the first stage, the LEC-γ1 binding assay of Example 4(A) wasutilized to pan the HL60 cDNA library and thereby to enrich for theplasmid of interest. Six μg of each HL60 cDNA library pool wasco-transfected with 2 μg of α3/4FT gene (Example 2) into COS cells.Approximately 45 hours post-transfection, the COS cells were lifted fromthe plates by incubating the cells in 1 mM EGTA for 15 min. at 37° C.,followed by scraping with cell lifters. The cells were washed twice inHanks buffered saline solution containing 1 mM calcium (HBSS). The cellswere resuspended in 4 ml of HBSS. The resuspended transfected COS cellswere screened using the LEC-γ1 binding assay described in Example 4(A).

The plasmids from adherent COS cells were recovered from a Hirts extract[B. Hirts, J. Mol. Biol., 26, 365-369 (1967)] and then electroporatedinto E. coli DH5α cells for amplification. The enriched population ofplasmids was purified over a CsCl gradient and re-transfected along withthe 3/4FT gene (Example 2) into COS cells. The transfection, screening,and plasmid amplification process was repeated for a total of threetimes before a pool that bound to the LEC-γ1-coated plates was visuallydetected. The positive plasmid pool was subsequently broken down intosubsets. This involved electroporating the Hirts extract from thepositive pool into E. coli DH5α cells and quantitating colonies per mlas described above. Various pool sizes were produced by plating out apredetermined number of colonies on agar plates in the presence ofampicillin. Duplicate plates were prepared by performing nitrocelluloselifts and storing the filters on new agar plates. The duplicate platesserved as reference plates for selecting individual or groups ofcolonies from any pool identified as being positive.

In the second stage of cloning, COS cells were co-transfected with thesublibrary pools and the 3/4FT gene by the same procedure used in theinitial steps of screening. Forty-eight hours post-transfection, thetransfected cells were screened using the fluorescent CHO:P-selectinassay of Example 4(B). Positive pools were further subdivided, asdescribed above, until finally individual colonies were screened andpositive clones identified. Using this method, a single positive clone,pMT21:PL85, was found to encode the P-selectin ligand protein. The DNAsequence of the P-selectin ligand contained in pMT21:PL85 is set forthin SEQ ID NO:1, and the binding characteristics of the P-selectin ligandprotein encoded by pMT21:PL85 are set forth in Example 4(C) below.

EXAMPLE 2 Cloning the α1,3/1,4 Fucosyltransferase Gene

The α1,3/1,4 fucosyltransferase gene (3/4FT) was cloned from total humangenomic DNA (Clontech Laboratories) by means of PCR. The senseoligonucleotide primer contained an XbaI site and the 5′ terminus of thegene (5′-TAGCATACGCTCTAGAGCATGGATCCCCTGGGTGCAGCCAAGC-3′, SEQ ID NO:9),and the antisense oligonucleotide primer contained an EcoRI site and the3′ terminus of the gene (5′-CCGGAATTCTCAGGTGAACCAAGCCGC-3′, SEQ IDNO:10). The PCR product was sequentially digested with XbaI and EcoRIand purified by standard gel purification methods. This gene was thenligated with vector pMT3 Sv2ADA (R. Kaufman, Methods in Enzymology,supra) that had also been sequentially digested with XbaI and EcoRI andpurified by standard gel purification methods. Competent HB11 cells(Biorad) were transformed with this ligation product and then plated onagar plates in the presence of ampicillin. Nitrocellulose filter liftsof ampicillin-resistant transformants were probed with a radiolabelledoligonucleotide (5′-AAGTATCTGTCCAGGGCTTCCAGGT-3′, SEQ ID NO:11)complementary to the nucleotide region 506-530 in the middle of the gene(J. Sambrook et al., supra).

Plasmid DNA minipreps were prepared from twelve positive clones. Thepurified DNA was then digested with EcoRI and XbaI to identify thecorrect clone with the proper size insert. This clone (pEA.3/4FT) wasthen grown up large scale and the DNA isolated by CsCl density gradientbanding (J. Sambrook et al., supra). DNA sequencing confirmed theidentity of the 3/4FT gene. The functionality of the gene was assessedin a cell-cell binding assay as follows. COS-1 monkey cells [(clone M6;M. Horwitz et al., Mol. Appl. Genet., 2:147-149, (1983)] weretransfected with 3/4FT using DEAE dextran followed by DMSO shocktreatment and chloroquine incubation [L. Sompeyrac and K Dana, Proc.Natl. Acad. Sci., 78:7575-7578 (1981); M. Lopata et al., Nucleic AcidsRes., 12:5707-5717, (1984); H. Luthman and G. Magnuson, Nucleic AcidsRes., 11:1295-1308, (1983)]. The transfected COS cells were suspendedand quantitated for binding to a CHO line expressing E-selectin [G.Larsen et al., J. Biol. Chem. 267:11104-11110, (1992)]. This assayconfirmed that the COS cells transfected with 3/4FT can express thesialylated Lewis^(x) epitope on the cell surface.

EXAMPLE 3 Expression of the P-Selectin Ligand Protein

A. Expression of the P-Selectin Ligand in LEC11 Cells

Functional P-selectin ligand was expressed in the SLe^(x)-positiveChinese hamster ovary (CHO) cell line LEC11 (Campbell, C. and Stanley,P. Cell 35:303-309 (1983) as follows: approximately 8 μg of plasmidcontaining the P-selectin ligand gene (pMT21:PL85, Example 1) wastransfected into LEC11 cells. At 68 hours post-transfection, the cellswere treated with 2.5 mM sodium butyrate for 4 hours. The cells wereobserved to induce P-selectin adhesion, as determined using the 6-CFDlabeled CHO:P-selectin cell binding assay (described in Example 4,section B). In contrast, neither LEC11 cells alone nor LEC11 cellstransfected with a control plasmid induced P-selectin adhesion.

B. Expression of Soluble P-Selectin Ligand in COS Cells

COS cells were transfected with 8 μg pED.sPSL.T7 (see Example 5C) and 4μg pEA.3/4 FT plasmid of Example 2, 8 μg pED.sPSL.T7 alone, or 8 μgplasmid vector (pMT21) and 4 μg pEA.3/4 FT gene. Forty-five hrpost-transfection, the cells were rinsed twice in PBS and incubatedovernight at 37° C. in serum-free DMEM minus phenol red (JRHBiosciences) supplemented with 2 mM L-glutamine, 100 U/ml penicillin and100 μg/ml streptomycin. Phenylmethylsulfonyl fluoride, aprotinin andNaN₃ were added to final concentrations of 1 mM, 2 μg/ml and 0.02%,respectively, and the conditioned medium was centrifuged to remove alldebris.

For immunoprecipitation experiments, the labeled soluble P-selectinligand protein was produced by co-transfecting COS cells withpED.sPSL.T7 and pEA.3/4 FT. At forty-five hr post-transfection, the COScells were labeled with 250 μCi/ml ³⁵S methionine (NEN) for 5 hours andthe medium was collected. Expression of sPSL.T7 protein was confirmed byimmunoprecipitation with anti-T7 antibodies.

C. Expression of PACE-Cleaved P-Selectin Ligand in COS Cells

COS cells were co-transfected with the pED.sPSL.T7 plasmid of Example5(C), the pEA.3/4FT cDNA of Example 2, and a plasmid containing the PACEcDNA as set forth in SEQ ID NO:5. A parallel control co-transfection wasdone using only the pED.sPSL.T7 plasmid and the pEA.3/4FT plasmid. After45 hours, conditioned medium from these transfected COS cells was coatedonto plastic dishes and binding to CHO:P-selectin cells (Example 4) wasdetermined. An approximately two-fold increase in bound CHO:P-selectincells was observed for dishes coated with medium containing theP-selectin ligand co-expressed with PACE, as compared with mediumcontaining P-selectin ligand which had not been co-expressed with PACE.Amino acid sequencing of the N-terminus of purified sPSL.T7 protein fromthe PACE co-transfection showed that all of the ligand had been cleavedat the PACE consensus site (amino acids 38-41 of SEQ ID NO:1).Radiolabelling of co-transfected COS cells with ³⁵S-methionine andsubsequent SDS-polyacrylamide gel electrophoresis and autoradiographyshowed that comparable quantities of the P-selectin ligand had beensecreted in both co-transfections.

D. Expression of the P-Selectin Ligand Protein in CHO Cells

A full-length form (amino acids 1-402) of the P-selectin ligand proteinwas expressed in the CHO(DUKX) cell line (Urlaub & Chasin, Proc. Natl.Acad. Sci. USA 77, 4216-4220 (1980)) as follows: approximately 25 μg ofthe pMT21:PL85 plasmid and approximately 8 μg of the pED.3/4FT (producedby restriction of pEA.3/4FT with EcoRI and XbaI and insertion of theresulting fragment into the pED plasmid) were co-transfected intoCHO(DUKX) cells using the calcium phosphate method. Transfectants wereselected for resistance to methotrexate. After two weeks, individualcolonies were screened for SLe^(x) expression by using a conjugate of ananti SLe^(x) antibody (CSLEX-1, U.S. Pat. No. 4,752,569) and sheep redblood cells (sRBC) prepared by the chromic chloride method (Goding, J.W., J. Immunol. Methods 10:61-66 (1976) as follows: sRBC were washedwith 0.15M NaCl until the wash became clear and then a 50% suspension ofsRBC was prepared in 0.15M Nacl. One ml of 0.01% chromic chloridesolution was added dropwise while vortexing to 0.2 ml of a sRBCsuspension containing 50 μg of CSLEX-1. After incubating at 37° C. for30 minutes, 10 ml of phosphate buffered saline (PBS) solution was addedto the reaction. The conjugate was washed once before resuspending into10 ml of PBS. The plates containing transfectants were washed with PBSand then 3 ml of PBS and one ml of the sRBC/CSLEX-1 conjugate was addedto each plate. Positive colonies were red on a transilluminator and werepicked into alpha medium with 10% fetal bovine serum. After two weeks,colonies were subjected to stepwise amplification using methotrexate atconcentrations of 2, 10, 25, 100, 250 nM. The stable cell line obtainedwas designated CD-PSGL-1 (R3.4). Expression of the P-selectin ligandprotein was confirmed by immunoprecipitation studies using thepolyclonal anti-P-selectin ligand protein antibody of Example 7(A). Thefunctionality of the P-selectin ligand protein produced by the CD-PSGL-1(R3.4) cell line was tested by assaying the transfectants for binding toLEC-γ1 as in Example 4(A).

The sPSL.T7 protein was expressed in a stable CHO-PACE line which wasalready expressing the cDNA encoding PACE as set forth in SEQ ID NO:5under adenosine deaminase selection (Kaufman, et al., PNAS (USA)83:3136-3140 (1986)). The psPSL.T7 (25 μg) and pED.3/4FT (8 μg) plasmidswere cotransfected into CHO-PACE cells using the calcium phosphatemethod. Transfectants were selected for resistance to methotrexate, andindividual colonies which bound to the sRBC/CSLEX-1 conjugate werepicked. After two weeks in culture, the colonies were subjected tostepwise amplification as described above. The stable cell line obtainedwas designated CP/PSL-T7 (R4.1). Expression of sPSL.T7 protein wasconfirmed by standard immunoprecipitation methods using either a T7specific monoclonal antibody or the LEC-γ1 chimera of Example 4(A). In asimilar fashion, a stable cell line expressing the mature full lengthform (amino acids 42-402) of the P-selectin ligand protein was obtainedby co-transfection of pMT21:PL85 and pED.314FT into the CHO-PACE line.

Stable cell lines expressing the sPSL.Q protein of Example 5(B) and thesPSL.Fc protein of Example 5(D) were constructed as follows: plasmidspED.sPSL.Q (25 μg) or pED.sPSL.Fc (25 μg) were cotransfected withapproximately 25 μg of the pED.3/4FT plasmid described above andapproximately 20 μg of a plasmid containing the PACE cDNA as set forthin SEQ ID NO:5) as well as the neomycin resistance gene into CHO(DUKX)cells using the calcium phosphate method. Transfectants were selectedfor resistance to methotrexate and the G418 antibiotic. Approximatelytwo weeks later, individual colonies were screened for SLe^(x)expression using sRBC/CSLEX-1 conjugate binding. The positive colonieswere picked in G418 medium at 1 mg/ml concentration. After 2-3 weeks inculture, cells were amplified with methotrexate in a stepwise selection.The stable cell lines obtained were designated CD-sPSL.Q (R8.2) andCD-sPSL.Fc (R8.1), respectively. The expression of sPSL.Q and sPSL.Fcprotein was confirmed by standard immunoprecipitation method using theanti P-selectin ligand protein polyclonal antibody of Example 7(A).

EXAMPLE 4 Assays of P-Selectin-Mediated Intercellular Adhesion

A. LEC-γ1 Binding Assay

A DNA encoding a chimeric form of P-selectin conjugated to the Fcportion of a human IgGγ1 (LEC-γ1) was constructed using known methods(Aruffo et al. Cell 67, 3544 (1991)), and stably transfected into dhfrCHO cells (CHO DUKX) for high level production of the chimeric LEC-γ1protein, which was purified for use in the binding assay set forthbelow.

Petri dishes were coated first with a polyclonal anti-human IgGγ1 Fcantibody and then with LEC-γ1. This method orients the LEC-γ1 constructsuch that the P-selectin portion of the chimeric molecule is presentedon the surface of the plates. Adhesion of HL60 cells to the orientedLEC-γ1 was quantitated in the presence and absence of calcium. HL60adhesion was shown to be calcium dependent, confirming that the chimericmolecule had retained functional binding of P-selectin to its ligand onHL60 cells. The binding of HL60 cells to oriented LEC-γ1 was also shownto be blocked by a neutralizing monoclonal antibody to P-selectin,demonstrating the specificity of P-selectin binding.

B. Fluorescent CHO-P-Selectin Binding Assay

The assay employed a fluorescently labeled CHO:P-selectin cell line(Larsen et al., J. Biol. Chem. 267, 11104-11110 (1992)) that can bind toand form clusters on the surface of COS cells that are co-transfectedwith the P-selectin ligand gene and the 3/4 FT gene. The CHO:P-selectincells were suspended at 1.5×10⁶ cells/ml in 1% fetal bovine serum in DMEmedium and labeled by adding 6-carboxyfluorescein diacetate (6-CFD) to afinal concentration of 100 ug/ml. After incubation at 37° C. for 15minutes, the cells were washed in medium and resuspended at 1×10⁵cells/ml. Five ml of the labeled cells were added to each washed COStransfectant-containing plate to be assayed and incubated at roomtemperature for 10 minutes. Nonadherent cells were removed by fourwashes with medium. The plates were then scanned by fluorescencemicroscopy for rosettes of adherent CHO:P-selectin cells.

C. Quantitative Adhesion Assay using Radioactively LabeledCHO:P-Selectin Cells

COS cells were co-transfected with the pMT21:PL85 plasmid of Example 1and the pEA.3/4FT plasmid of Example 2 by the same procedure used in theinitial stages of screening. As controls, COS cells were transfectedwith pMT21:PL85 alone, or with pEA.3/4FT alone, or with a similarplasmid containing no insert (“mock”). 24 hours post-transfection, thetransfected cells were trypsinized and distributed into Costar 6-welltissue culture plates. CHO:P-selectin cells were labeled for 16 hourswith ³H-thymidine using known methods and preincubated at 0.5×10⁶cells/ml for 30 minutes at 4° C. in α medium containing 1% BSA(control); α medium containing 1% BSA, 5 mM EDTA and 5 mM EGTA; α mediumcontaining 1% BSA and 10 μg/ml of a neutralizing anti P-selectinmonoclonal antibody; and α medium containing 1% BSA and anon-neutralizing anti-P-selectin monoclonal antibody. The preincubatedcells were then added to the wells containing the transfected COS cells.After a 10 minute incubation, unbound cells were removed by 4 changes ofmedium. The bound CHO:P-selectin cells were released by trypsinizationand quantified by scintillation counting.

COS cells co-transfected with P-selectin ligand and the 3/4FT inducedapproximately 5.4-fold more binding of CHO:P-selectin cells relative toCOS mock cells; assay in the presence of EGTA and EDTA reduced bindingto the level of the mock transfected COS cells. Likewise, incubationwith neutralizing anti-P-selectin antibody also eliminated specificbinding, whereas non-neutralizing antibody had no effect. In contrast,the binding of CHO:P-selectin to COS cells transfected with P-selectinligand alone was not statistically different than binding to themock-transfected COS in both the presence or absence of EDTA and EGTA,or anti-P-selectin antibodies. The binding of CHO:P-selectin cells toCOS cells transfected with 3/4 FT alone was approximately 2-fold greaterthan to the mock-transfected COS, but was unaffected by the presence orabsence of EDTA and EGTA.

EXAMPLE 5 Construction of Soluble P-Selectin Ligands

The EcoRI adaptors used to generate the cDNA library from HL60 cells inExample I contain an XbaI restriction site (TCTAGA) just 5′ of thebeginning of SEQ ID NO:1 as it is located in the pMT21:PL85 plasmid. Inorder to generate soluble forms of the PSL, the pMT21:PL85 plasmid wasrestricted with XbaI and with HincII (which cleaves after nucleotide 944of SEQ ID NO:1). The approximately 950 bp fragment thus generated,containing all of the encoded extracellular segment of the ligand up toand including the codon for valine 295, was isolated and used togenerate DNAs encoding soluble forms of the P-selectin ligand protein asset forth in sections A though D below.

A. Construction of psPSL.QC

The fragment was purified and ligated into mammalian expression vectorpED between the XbaI and EcoRI sites, along with double strandedsynthetic oligonucleotide DNA that recreated the codons from Asn 296 toCys 310 and introduced a novel stop codon immediately following Cys 310.The sequence of the oligos is as follows:

(SEQ ID NO: 12) 5′-AACTACCCAGTGGGAGCACCAGACCACATCTCTGTGAAGCAGTGCTAG(SEQ ID NO: 13) 5′-AATTCTAGCACTGCTTCACAGAGATGTGGTCTGGTGCTCCCACTGGG TAGTTThe resulting plasmid was designated pED.sPSL.QC, and the proteinexpressed from the plasmid was designated sPSL.QC.B. Construction of psPSL.Q

The fragment was purified and ligated into the pED plasmid (Kaufman etal., 1991) between the XbaI and EcoRI sites, along with the doublestranded synthetic oligonucleotide DNA that recreated the codons fromAsn 296 to Gln 309 and introduced a novel stop codon immediatelyfollowing Gln 309. The sequence of the oligos is as follows:

(SEQ ID NO: 14) 5′-AACTACCCAGTGGGAGCACCAGACCACATCTCTGTGAAGCAGTAG(SEQ ID NO: 15) 5′-AATTCTACTGCTTCACAGAGATGTGGTCTGGTGCTCCCACTGGGTAG TT The resulting plasmid was designated pED.sPSL.Q, and the proteinexpressed from the plasmid was designated sPSL.Q.C. Construction of psPSL.T7

Oligonucleotides encoding 14 amino acids including an epitope derivedfrom the phage T7 major capsid protein were synthesized, creating aC-terminal fusion of the epitope “tag” with an additional 32 amino acidsderived from the vector sequence. Two oligonucleotides having thesequences

(SEQ ID NO: 16) 5′-CTAGACCCGGGATGGCATCCATGACAGGAGGACAACAAATGGTAGGC CGTAGand (SEQ ID NO: 17) 5′-AATTCTACGGCCTACCCATTTGTTGTCCTCCTGTCATGGATGCCATCCCGGGTwere duplexed and ligated with the large XbaI-EcoRI fragment ofmammalian expression plasmid pED. The resulting plasmid, pED.T7 wasrestricted with XbaI and SmaI and ligated to the 950 bp XbaI-HincIIfragment described above, resulting in plasmid pED.sPSL.T7. The proteinresulting from expression of pED.sPSL.T7 was designated sPSL.T7.D. Construction of Soluble P-selectin Ligand-IgGFc Chimera

The plasmid DNA encoding a soluble, extracellular form of the P-selectinligand protein fused to the Fc portion of human immunoglobulin IgG1 wasconstructed as follows: the mammalian expression vector pED.Fc containssequences encoding the Fc region of a human IgG1 with a novel linkersequence enabling the fusion of coding sequences amino terminal to thehinge region via a unique XbaI restriction site. A three fragmentligation was performed: pED.Fc was restricted with XbaI and gel purifiedin linear form. The 950 bp fragment from pMT21:PL85 described abovecomprised the second fragment. The third fragment consisted of annealedsynthetic oligonucleotide DNAs having the following sequence:

5′-CTGCGGCCGCAGT (SEQ ID NO: 18) 5′-CTAGACTGCGGCCGCAG (SEQ ID NO: 19)The ligation products were grown as plasmid DNAs and individual cloneshaving the correct configuration were identified by DNA sequencing. Theplasmid was designated pED.PSL.Fc. The DNA coding region of theresulting soluble P-selectin ligand IFc fusion protein is shown in SEQID NO:6.

EXAMPLE 6 Characterization of Expressed P-Selectin Ligands

A. Binding Characterization of Full-Length P-Selectin Ligand ProteinExpressed on COS Cells

Co-transfection of COS cells with the pEA.3/4FT plasmid of Example 2 andthe pMT21:PL85 plasmid of Example 1 yields COS cells which specificallybind to CHO:P-selectin cells. This binding is observed only uponco-transfection of pEA.3/4FT and pMT21:PL85; use of either plasmid alonegenerates COS cells which do not bind to CHO:P-selectin cells. Nobinding is observed between the parental CHO(DUKX) cell line which doesnot express P-selectin and COS cells co-transfected with pEA.3/4FT andpMT21:PL85. The binding between the co-transfected COS cells andCHO:P-selectin cells is sensitive to chelators of divalent ions such asEDTA and EGTA, consistent with the Ca⁺⁺ dependency of P-selectinmediated cellular adhesion. A neutralizing anti-P-selectin monoclonalantibody blocked the binding between the CHO:P-selectin cells and theCOS cells which had been co-transfected with pEA.3/4FT and pMT21:PL85,while a non-neutralizing anti-P-selectin monoclonal antibody had noeffect on the binding. The antibody results indicate that the functionaldomain(s) of P-selectin are required for binding to P-selectin ligandprotein expressed on the surface of COS cells.

B. Electrophoretic Characterization of Full-Length P-selectin LigandExpressed in COS Cells

Detergent extracts of co-transfected COS cells were prepared as follows:45 hours post co-transfection, approximately 1.5×10⁷ cells weresuspended in 5 ml of lysis buffer (10 mMpiperazine-N,N′-bis[2-ethanesulfonic acid] (PIPES) pH 7.5, 100 mM KCl, 3mM MgCl₂, 1 mM benzamidine, 0.5 μg/ml leupeptin, 0.75 μg/ml pepstatin, 1mM ethylmaleimide, and 1 μg/ml aprotinin) and lysed by sonication.Cellular debris was removed by low speed centrifugation (500×g. 10minutes), and a membrane fraction collected by ultracentrifugation(100,000×g, 60 min). The high speed membrane pellet was resuspended inan extraction buffer (10 mM 3-[N-Morpholino]propanesulfonic acid] (MOPS)pH 7.5, 0.1 M NaCl, 0.02% NaN₃, 1% Thesit® (Sigma), 1 mM benzamidine,0.5 μg/ml leupeptin, 0.75 μg/ml pepstatin, 1 mM ethylmaleimide, and 1μg/ml aprotinin). Samples were then subjected to SDS polyacrylamide gelelectrophoresis and transfer to nitrocellulose blots as follows: analiquot of the detergent extract was suspended in 1% SDS loading bufferand heated for 5 minutes at 100° C. before loading onto an 8-16%polyacrylamide gel (reduced) or a 6% gel (non-reduced) andelectrophoresed in the Laemmli buffer system. Blots were prepared usingImmobilon-P® transfer membranes. The blots were immersed in 10 mM MOPSpH 7.5, 0.1 M NaCl, 0.02% NaN₃, 1 mM MgCl₂, 1 mM CaCl₂, and 10% non-fatmilk overnight at 4° C. Blots were rinsed once in the above buffer,minus the milk, and incubated in blotting buffer (10 mMMOPS pH 7.5, 0.1MNaCl, 1% bovine serum albumin, 0.05% Thesit, 1 mM MgCl₂, 1 mM CaCl₂) for30 minutes at room temperature.

The blots were then probed for the P-selectin ligand as follows: 50 ngof a P-selectin/Fc chimera was pre-incubated with 3 μCi of ¹²⁵I-ProteinA in blotting buffer for 30 minutes at room temperature. Additionalexcipients (e.g., EDTA, EGTA, monoclonal antibodies) could be added tothe pre-incubation mixture at this point to evaluate their effects onbinding of the chimera to the P-selectin ligand. The pre-incubatedmixture was then incubated with the blots (prepared as above) for 60minutes at room temperature, and the blots were subsequently washed fourtimes with the same blotting buffer (without bovine serum albumin), airdried, and autoradiographed at −70° C.

Under non-reducing conditions, two bands were observed with thistechnique for membrane extracts prepared from co-transfected COS cells.The major band migrated with an estimated molecular weight ofapproximately 220 kD, whereas the minor band migrated with a molecularweight of approximately 110 kD. Under reducing conditions, only a singleband was observed with a molecular weight of approximately 110 kD,indicating that under non-reducing conditions, the P-selectin ligandexists as a homodimer. The approximate molecular weight of the reducedmonomer is greater than that predicted from the deduced amino acidsequence of the cDNA clone (45 kD), indicating that the expressedprotein undergoes extensive post-translational modification (see Example6(C)). The specificity of the P-selectin/Fc chimera was confirmed by theobservation that a nonspecific IgG₁ probe yielded no bands on the blots.Additionally, the binding of the P-selectin/Fc chimera to the blots wasabolished by EDTA, EGTA, and a neutralizing anti-P-selectin monoclonalantibody. Specific bands on the blots were observed only from membraneextracts of COS cells co-transfected with the pEA.314FT and pMT21:PL85plasmids. Membrane extracts from control transfections (pEA.3/4FT orpMT21:PL85 alone) failed to yield observable bands on blots.

C. Glycosylation of P-selectin Ligand Protein

The presence of covalently attached carbohydrate on recombinantP-selectin ligand and its role in binding to P-selectin was determinedas follows: COS cells were co-transfected with pED.sPSL.T7 of Example5(C) and the pEA.3/4FT plasmid of Example 2. After 48 hours, the cellswere pulsed with ³⁵S-methionine. 200 μl of ³⁵S methionine-labeledsPSL.T7 conditioned medium was incubated with 5 μg LEC-γ1 in thepresence of 2 nM CaCl₂ and 1 mg/ml bovine serum albumin (BSA). Afterrotating for 2 hours at 4° C., Protein A-Sepharose beads (Pharmacia)were added for 1 hour at 4° C., pelleted by centrifugation and washedtwice in Tris buffered saline (20 mM Tris-HCl, 150 mM NaCl pH 7.5,hereinafter TBS) containing 2 mM CaCl₂ and 1 mg/ml BSA. The pellets werethen resuspended and treated with neuraminidase (Streptococcuspneumoniae), O-glycanase, and N-glycanase (all from Genzyme) as follows.All glycosidase digestions were done at 37° C. overnight. Forneuraminidase digestion, the pellet was resuspended in 50 μl2-(N-morpholino)-ethanesulfonic acid (MES) buffer, pH 6.5 (Calbiochem)and 0.1% SDS, heated at 95° C. for 5 minutes, then pelleted. Thesupernatant was modified to contain 1.4% n-Octyl B-D-glucopyranoside(OGP), 10 mM calcium acetate, 20 mM sodium cacodylate and 2.5 mM PMSF,final pH 7.0 Eight μl neuraminidase was added for a final concentrationof 1 unit/ml. For neuraminidase/O-glycanase digestion, the sample wasprepared as above and along with the neuraminidase, the O-glycanase wasalso added to a final concentration of 0.1 unit/ml. For N-glycanasedigestion, the pellet was resuspended in 54 ul MES buffer and 1% SDS,heated at 95° C. for 5 minutes, then pelleted. The supernatant wasmodified to contain 0.2 M sodium phosphate, 3.5% OGP, and 2.5 mM PMSF,final pH 8.5. N-glycanase was added for a final concentration of 12units/ml and incubated as above.

The effect of glycosidase treatment on sPSL.T7 was assessed in two ways.For this, each digested protein sample was divided into two equalfractions. One fraction was precipitated with the P-selectin polyclonalantibody of Example 7(A), to show the effect of digestion on theelectrophoretic mobility. The other fraction was precipitated with theLEC-γ1 chimera of Example 4(A), to assess the remaining P-selectinligand binding activity after digestion. The immunoprecipitationedsamples were analyzed by SDS-polyacrylamide gel electrophoresis underreducing conditions and autoradiography.

In the absence of glycosidase treatment, autoradiography revealedcomparable bands (with molecular weights of 110 kD) for eachprecipitation. When the P-selectin ligand protein was treated withneuraminidase, anti-P-selectin ligand polyclonal antibody precipitationrevealed a slight decrease in mobility, consistent with removal ofsialic acid residues. The amount of P-selectin ligand proteinprecipitated by LEC-γ1 was significantly reduced after neuraminidasetreatment, consistent with the role of sialic acid residues in theP-selectin/P-selectin ligand interaction. When the P-selectin ligandprotein was treated with both neuraminidase and O-glycanase, asubstantial increase in electrophoretic mobility was observed afterprecipitation with the anti-P-selectin ligand polyclonal antibody,indicating that a number of O-linked oligosaccharide chains had beenremoved. However, removal of O-linked oligosaccharides from theP-selectin ligand protein may not have been complete, since theelectrophoretic mobility did not correspond to a protein with amolecular weight of 38 kD, as would be predicted from the amino acidsequence set forth in SEQ ID NO:1. The neuraminidase/O-glycanasedigested P-selectin ligand protein bound to LEC-γ1 very poorly, furtherindicating the role of oligosaccharides in the P-selectin/P-selectinligand interaction. Treatment of the purified P-selectin ligand withN-glycanase resulted in a slight increase in electrophoretic mobility,demonstrating that some of the consensus sites for N-linkedglycosylation are occupied. The amount of P-selectin ligand proteinprecipitated by LEC-γ1 was slightly reduced, indicating that N-linkedglycosylation also contributes to the P-selectin/P-selectin ligandinteraction, though not as dramatically as sialyation and O-linkedglycosylation.

EXAMPLE 7 Polyclonal Antibodies Specific for P-Selectin Ligands

A. Polyclonal Rabbit Anti-P-Selectin Ligand Protein/Maltose BindingProtein Fusion Protein

The anti-P-selectin ligand polyclonal antibody was generated byimmunizing rabbits with a fusion protein generated in E. coli. Thefusion protein consisted of the amino terminal one-third of theP-selectin ligand (amino acids 1 to 110 of SEQ ID NO:1) fused in frameto the maltose binding protein (Maina, C. V. et al., Gene 74, 365-373(1988); Riggs, P., in Current Protocols in Molecular Biology F. M.Ausebel et al., Eds., Greene Associates/Wiley Interscience (New York,1990) chapter 16.6). Under conditions employed herein, the fusionprotein antibody recognizes the P-selectin ligand protein.

B. Polyclonal Rabbit Anti-sPSL.T7 Protein

A soluble form of the invention (sPSL.T7; see example 5(C)) was purifiedto apparent homogeneity according to the following scheme: COS cellswere transfected with three plasmids, one encoding each of thefollowing: sPSL.T7 (Example 5(C)), 3/4FT (Example 2), and a soluble formof PACE (as set forth in SEQ ID NO:5). After 72 hours, the conditionedmedium was collected and recombinant sPSL.T7 was purified as follows.

Conditioned medium was diluted two fold with 50 mM MOPS, 150 mM NaCl,0.5 mM CaCl₂ and 0.5 mM MnCl₂, pH 7.2, and applied to a column of lentillectin-Sepharose 4B equilibrated in the same buffer. After loading, thecolumn was washed with the same buffer until the optical absorbance at280 nm dropped to a stable baseline. The column was then eluted with thesame buffer which had been adjusted to 0.5 M α-methyl-mannoside and 0.3M NaCl. Recombinant sPSL.T7 was collected over 5-15 column volumes ofthis elution buffer. The lentil lectin eluate was then subjected to a0-70% ammonium sulfate precipitation by adding 472 g of ammonium sulfateper liter of column eluate at 4° C. After stirring for 30 minutes, theprecipitate was resuspended in a minimal volume of TBS (20 mM Tris-HCl,150 mM NaCl, pH 7.5) and applied to a TSK G4000SW_(XL) gel filtrationcolumn equilibrated in TBS. The flow rate on the column was 0.5 m/minand a guard column was employed. In aliquots of <250 μl, the resuspendedammonium sulfate pellet was injected on the column and fractionsanalyzed by SDS-PAGE with Western analysis. Fractions containing sPLS.T7were pooled and then used for immunizing rabbits.

Antibodies to sPSL.T7 were generated in the standard fashion by antigenpriming and subsequent boosting over a 3 month period. Specifically,primary immunization was performed by mixing 50 μg of sPSL.T7 (denaturedby mixing in 0.1% SDS and heating for 10 minutes at 100° C.) withcomplete Freund's adjuvant and injected at five sites subcutaneously.The second (and all subsequent) boosts were performed by mixing 25 μg ofsPSL.T7 (denatured by mixing in 0.1% SDS and heating for 10 minutes at100° C.) [12.5 μg for the third and subsequent boosts] with incompleteFreund's adjuvant and injecting at two sites subcutaneously (or later,intramuscularly) every two weeks. Test bleeds were performed every twoweeks to monitor antibody titer. When the antibody titer reached asuitable level, a larger scale bleed was performed and a total serumfraction prepared. This polyclonal antibody preparation was used toinhibit the specific binding of HL60 cells to CHO:P-selectin cells in amanner similar to that described in Example 4.

This assay employed fluorescently-labeled HL60 cells (labelled withBCECFAM; 2′,7′-bis-(2-carboxymethyl)-5-and-6)-carboxyfluorescein,acetoxymethyl ester) binding to CHO cells plated on the bottom ofmicrotiter plates. The labelled HL60 cells were pre-incubated witheither sera containing polyclonal antibody or with pre-immune sera for30 minutes at 4° C. The cells were then washed and incubated with theCHO:P-selectin cells for 10 minutes. The plates were then washed and thefluorescence read with a fluorescence microtiter plate reader. Usingthis assay, a 1:15 dilution of the anti-sPSLT7 polyclonal serum resultedin essentially complete inhibition of HL60 cell binding toCHO:P-selectin. Demonstrable inhibition of HL60 binding toCHO:P-selectin was still observed at antiserum dilutions of 1:150.Pre-immune serum had no effect on HL60 cell binding to CHO:P-selectin.

EXAMPLE 8 Cotransformation with Core2

A. Isolation of the cDNA Encoding Core2 GlcNAc Transferase

The cDNA encoding core2 GlcNAc transferase was isolated by standardmolecular biology techniques. Two oligos were designed at the 5′ and 3′end (including translational initiation and termination codon,respectively) based on the published human core2 sequence (Bierhuizen,M. F. A., Fukuda, M., Proc. Natl. Acad. Sci. 89, 9326-9330 (1992)). Thepools of an HL60 cDNA library (Sako, D., Cell 75, 1179-1186 (1993)) wereused as template to amplify the core2 coding sequence by a standard PCRprotocol. The PCR amplified fragment was purified and subcloned into pEDvector. To isolate cDNA, the pools which gave a positive signal in thePCR reaction were transformed into E. coli and plated. Transformantswere transferred onto nitrocellulose filters and hybridized with a ³²Pradiolabelled PCR fragment according to standard protocols. Positiveclones were picked and purified by replating. The sequence of the cDNAand PCR clone was confirmed by dideoxy sequencing.

B. Generation of Stable PSGL-1 Chinese Hamster Ovary Cell LinesExpressing Core2 Enzyme

A cell line made in accordance with the methods of Example 3 expressingfull-length P-selectin ligand protein and 3/4 fucosyltransferase wasco-transfected with core2 cDNA and a neomycin resistance gene (pMT4Neo)by standard calcium phosphate methods. After about two weeks, stableG418-resistant transfectants were picked either as single isolates or ina pool. These transfectants were grown in 1 mg/ml G418 complete DMEMmedia and analyzed for core2 enzyme activity (Higgins, E. A., et al., J.Biol. Chem. 266, 6280-6290 (1991)). Positive clones or pools foundpositive for core2 activity were analyzed for P-selectin ligand bindingto P-selectin by various methods. In a similar fashion, cell linesexpressing either P-selectin ligand protein or soluble P-selectin ligandprotein with both the 3/4 fucosyltransferase and PACE enzymes (seeExample 3) were used to isolate stable cotransfectants of core2 asdescribed above.

C. Effects of Core2 on P-Selectin Binding Activity

The effects of core2 on P-selectin binding activity was evaluated bythree different methods:

1. Binding of mPSGL-1 Transfectants to Immobilized Soluble P-Selectin orP-Selectin/IgG Chimera.

48-well plates were coated with 1 ug/ml anti human Fc antibody in 50 mMTris pH 9.5 at 4° C. for five to six hours. After washing twice withHBSS buffer, P-selectin/IgG chimera (0.1-1 ug/ml conc., Example 5) wasplated in HBSS buffer overnight at 4° C. The plates were blocked withBSA (3 mg/ml) at 4° C. for three to four hours. In the case of solubleP-selectin ligand protein, the protein was coated directly onto platesin the same buffer. The ³H labelled CHO cells were lifted with 2 mMEGTA, washed three times with PBS, and resuspended to a final density of10⁶ cells/ml. A 300 ul aliquot of this suspension was added to each well(300,000 cell/well). After incubating for 12 minutes at roomtemperature, wells were washed four times with serum free DMEM to removeunbound cells. Bound cells were lifted with 5 mM EGTA and counted inscintillation counter. U937 cells, used as a positive control for nativeP-selectin ligand protein binding, were pretreated with gamma globulin(5 mg/ml) to block endogenous Fc receptor before binding to P-selectinIgG chimera. Comparative binding data are shown in FIG. 1.

2. Immunoprecipitation of PSGL-1 with P-Selectin/IgG Chimera.

Recombinant full-length or soluble P-selectin ligand protein preparedfrom transformants, with and without additional core2, was labelled with³⁵S-methionine and subsequently immunoprecipitated with either the antiP-selectin ligand protein polyclonal antibody or P-selectin ligand/IgGchimera as described previously in Examples 7 and 5; Sako, D., Cell 175,1179-1186 (1993). Data are depicted in FIG. 2.

3. Flow Cytometry.

Stable murine P-selectin ligand protein transfectants (with and withoutcore2) were analyzed by standard FACS techniques using eitherP-selectin/IgG chimera (LecY1) (Example 5) or anti P-selectin ligandprotein monoclonal antibody (MAb 275, raised against a peptide havingthe sequence from amino acid 42 to amino acid 56 of SEQ ID NO:2). Bothreagents were preconjugated to FITC labelled Protein A. Cells wereanalyzed by FACS after incubating with this conjugate for 30 minutes at4° C. in the presence of 2 mM CaCl₂. Data are depicted in FIG. 3.

EXAMPLE 9 E-Selectin Binding of P-Selectin Binding Protein

E-Selectin/IgG chimera was made as described in Example 5 for theP-selectin IgG chimera using an E-selectin encoding DNA including aminoacids-21 to 536 of the sequence reported in Bevilacqua et al., Science,243:1160 (1989).

U937 cells (approximately 6.5×10⁷) were recovered from tissue cultureplates and divided equally into two 50 mL cultures (final concentrationof 1.3×10⁶ cells/mL) containing fresh complete RPMI medium and 50 μCi/mlof ³H-glucosamine hydrochloride (labels the protein-linked carbohydrateof glycoproteins [Varki, FASEB 5:226-235 (1991)]. After 48 hoursincubation, the cells from both cultures were recovered bycentrifugation and washed three times with PBS. The pelleted cells weresuspended in 2.5 mL each of a lysis buffer containing 1% Triton X-100and disrupted by probe sonication for two minutes. The detergent lysateswere placed on ice for three hours and then resonicated for anadditional two minutes. The lysates were centrifuged at 16,000 rpm forfive minutes, the supernatants were recovered and each adjusted to 12 mLwith lysis buffer containing no detergent. To one of the two dilutedcell lysates was added 100 uL of protein A sepharose precoupled withP-selectin/IgG chimera (see Example 5) and to the other was added 100 uLof protein A sepharose precoupled with E-selectin/IgG chimera. Bothchimeric proteins were present at a density of approximately 2 mgprotein/mL of resin. Binding reactions were allowed to proceed overnightat 4 degrees C. with end-over-end mixing. On occasion, purifiedmembranes from U937 cells served as the starting material for thedetergent extraction of labeled proteins. In these cases, the detergentextraction and affinity precipitation steps were essentially identicalto the above.

Following incubation, the two parallel reaction mixtures were eachcentrifuged at 2,000 rpm and supernatants were discarded. The resinpellets were washed four times with buffer (10 mM MOPS, 100 mM NaCl, 1mM CaCl₂, 1 mM MgCl₂, 0.02% NaN₃, pH 7.5 with Triton X-100 [0.25% forthe first and second washes, 0.1% for the third wash and 0.01% for thefourth wash]). A final 1 mL pre-elution wash of each resin pellet usingbuffer containing 0.01% Triton X-100 was conducted and these wereretained for quantitation of radioactive counts by liquid scintillationcounting (LSC). The resins were then eluted overnight at 4 degrees C.with end-over-end mixing in 1 mL each of buffer containing 0.01% TritonX-100 and 10 mM EDTA. The supernatants were recovered by centrifugationand then quantitated by LSC.

Autoradiography of the materials released from the resins by EDTA wasperformed by electrophoresis of samples (approximately 10,000 cpmsamples concentrated by Centricon-10 units where needed) on 10%cross-linked SDS-PAGE gels, subsequent treatment of the gels withEN3HANCE (Dupont) as per the manufacturer's instructions followed bydrying for two hours on a commercially available gel dryer (Bio-Rad).Exposure of the dried gels to X-ray film was conducted for a minimum ofthree days at −80 degrees C.

Elution of immobilized E- or P-selectin, previously exposed to detergentextracts of U937 cells and exhaustively washed, with EDTA yieldedliberated, ³H-glucosamine labeled proteins. The amount of radiolabelrecovered from the EDTA eluates was at least 10-fold higher than thecounts observed in the final, pre-EDTA washes. This observation suggeststhat both P- and E-selectin chimeras affinity captured ligand(s) fromU937 whole cell lysates in an EDTA-dependent manner and that capturedligands were subsequently released upon treatment of the resins withEDTA.

The evaluation of the proteins released by EDTA from the two chimeraswas performed by SDS-PAGE and autoradiography under reducing andnon-reducing conditions (commercially available ¹⁴C-labeled molecularweight standards were employed). As shown by the autoradiograph depictedin FIG. 4, the released counts from the whole cell lysates treated withthe P-selectin chimera (lanes 2 and 10) and the E-selectin chimera(lanes 4 and 8) correlated to a major species of 200 kD molecularweight, non-reduced (lanes 2 and 4), and 100 kD reduced (lanes 8 and10). In different experiments depicted in FIG. 4, where purifiedmembrane extracts were used as the starting material in place of wholecells, both the E-selectin chimera (lane 3, non-reduced and lane 9,reduced) and the P-selectin chimera (not shown) gave similar results.Other experiments have demonstrated that the major U937 glycoproteinwhich binds to P-selectin is immunoreactive with Rb3026, a polyclonalantibody raised against recombinant sPSGL1.T7. Therefore, P- andE-selectin specifically recognize a single major glycoprotein specieswith identical properties in each case.

EXAMPLE 10 Production and Analysis of Deleted or Altered Forms ofSoluble P-Selectin Ligand Protein

A. Generation of DNA Constructs

Truncated forms of the P-selectin ligand protein-IgG chimeras weregenerated as follows. Plasmid pED.PSL.Fc was restricted with PstI andNotI and the 6 kb fragment comprising the Fc portion and vector, pEDFc6kb, was gel purified. Plasmid constructs pED. 149.Fc, pED.47.Fc and pED.19.Fc were created by standard PCR technique, using the following pairsof oligonucleotide primers:

“Upstream” primer for all constructs:

(SEQ ID NO: 20) 5′-CCAGGTCCAACTGCAGGTCGACTCTAGAGGGCACTTCTTCTGGGCCCACG-3′

“Downstream” primer for 148Fc:

(SEQ ID NO: 21) 5′-TATTATCTGTGCGGCCGCCCTCCAGAACCCATGGCTGCTGGTTGCAGTGG-3′

“Downstream” primer for 47Fc:

(SEQ ID NO: 22) 5′-TATTATCTGTGCGGCCGCGCAGCAGGCTCCACAGTGGTAG-3′

“Downstream” primer for 19Fc:

(SEQ ID NO: 23) 5′-TATTATCTGTGCGGCCGCGGAGGCTCCGTTTCTGGCAG-3′.The template DNA for PCR reaction was pED.PSL.Fc. The PCR conditionswere 94° C., 1 min.; 42° C., 1 min.; 72° C., 3 min.; 25 cycles, using aPerkin-Elmer Thermocycler. After completion of the last cycle, thereaction was treated with Klenow enzyme at 25° C. for 30 min., extractedwith phenol chloroform, sodium acetate added to 0.3M, and the PCRproduct DNA was precipitated with 2.5 volumes of ethanol. The DNA pelletwas rinsed with 70% ethanol and residual ethanol was evaporated. Theresuspended DNA was digested with PstI and NotI, gel purified andligated with the pEDFc6 kb fragment described above. Correct constructswere identified by restriction analysis and confirmed by DNA sequencing.

Plasmid pED.ΔY148.Fc, pED.H24.Q70.148.Fc were created by site directedmutagenesis (Maniatis et al., 1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratories) using pED.148Fc as template andthe following mutagenesis oligonucleotides:

for ΔY148: (SEQ ID NO: 24) 5′-CGGAGACAGGCCACCGAATTCCTGCCAGAAACG-3′for H24: (SEQ ID NO: 25) 5′-CCTCCAGAAATGCTGAGGCACAGCACTGACACCACTCCTC-3′for Q70: (SEQ ID NO: 26) 5′-GAGCTGGCCAACATGGGGCAACTGTCCACGGATTCAGCAG-3′Positive clones were identified by colony hybridization (Maniatis et al,supra).

pED.FFFE.148.Fc was constructed by restricting pED.AY148.Fc with EcoRIand ligating the following duplexed oligonucleotides:

5′-AATTCGAGTTCCTAGATTTTG-3′ (SEQ ID NO: 27) and5′-AATTCAAAATCTAGGAACTCG-3′. (SEQ ID NO: 28)

Constructs of the series pED.FYYD.19.Fc, pED.FFYD.19.Fc andpED.FFFD.19.Fc, were made by restricting pED.ΔY148.Fc with EcoRI andNotI and ligating the following duplexed oligonucleotides:

for pED.FYYD.19.Fc:

(SEQ ID NO: 29) 5′-AATTCGAGTACCTAGATTATGATTTCCTGCCAGAAACTGAGCCTCCG C-3′and

(SEQ ID NO: 30) 5′-GGCCGCGGAGGCTCAGTTTCTGGCAGGAAATCATAATCTAGGTACTC G-3′;for pED.FFYD.19.Fc:

(SEQ ID NO: 31) 5′-AATTCGAGTTCCTAGATTATGATTTCCTGCCAGAAACTGAGCCTCCG C-3′and

(SEQ ID NO: 32) 5′-GGCCGCGGAGGCTCAGTTTCTGGCAGGAAATCATAATCTAGGAACTC G-3′;for pED.FFFD.19.Fc:

(SEQ ID NO: 33) 5′-AATTCGAGTTCCTAGATTTCGATTTCCTGCCAGAAACTGAGCCTCCG C-3′and

(SEQ ID NO: 34) 5′-GGCCGCGGAGGCTCAGTTTCTGGCAGGAAATCGAAATCTAGGAACTC G-3′.B. Plate Binding Assay for Analysis of Deleted or Altered Forms ofSoluble P-Selectin Ligand Protein

The individual plasmid DNAs encoding the various mutated forms ofsoluble PSGL-1/Fc chimeras were co-transfected with pEA.3/4FT and PACEcDNA in COS cells as described in Example 3(c). 50 mls of serum freemedium, collected 40-64 hours post transfection from approximately 10⁷COS cells, was purified on a column of 0.25 ml of protein A sepharose(Pharmacia) equilibrated with TBS supplemented with 2 mM CaCl₂. Afterwashing with 20 mls of TBS/CaCl₂, the bound material was eluted with 0.5mls of 0.1M acetic acid, 0.15M NaCl, 2 mMCaCl₂. The eluted material wasneutralized with 1/20th volume 3M Tris pH 9.0. The material wasquantitated by measuring absorbance at 280 nm and by comassie bluestaining of PAGE/SDS/Laemmli gels.

In order to produce non-sulfated forms of soluble PSGL-1, COS celltransfections of the relevant Fc chimeras were performed as describedabove except that following transfection the cells were cultured in thepresence of 50 mM Chlorate (Sigma).

Quantitative adhesion of CHO:P-selectin, CHO:E-selectin andCHO:L-selectin expressing cells was performed as described in Example4(c), with the following modifications: COS cell and antibodies wereomitted. Instead, 48-well microtiter plates (Costar) were coated for 16hours at 4° C. with varying quantities of protein A-purified solublePSGL-1/Fc chimeras. The unbound material was removed and the coatedwells were treated with Hank's buffered saline (HBS) with 1 mg/ml BSAand 2 mM CaCl₂ for 1 hour at 4° C. Tritium labeled CHO selectinexpressing cells were added and binding quantitated as described inExample 4(c).

C. Effects of Alteration of N-Linked Glycosylation Sites

Constructs expressing three P-selectin ligand-IgG chimeras wereconstructed to examine the effects of N-linked glycosylation sites onselectin binding. These constructs had the following characteristics:

-   -   148.Fc amino acids 42-189 of SEQ ID NO:2    -   Q70.148.Fc amino acids 42-189 of SEQ ID NO:2, with the        asparagine residue at position 111 of SEQ ID NO:2 replaced with        a glutamine residue    -   H24.Q70.148.Fc amino acids 42-189 of SEQ ID NO:2, with the        asparagine residue at position 65 of SEQ ID NO:2 with a        histidine residue and the asparagine residue at position 111 of        SEQ ID NO:2 replaced with glutamine residue        These constructs are schematically represented in FIG. 6.

The binding of these constructs to protein A and P-selectin-IgG chimera(LEC-γ1) was compared. The results of these experiments are shown inFIG. 7. Comparison of lanes 4, 5 and 6 in the autoradiographdemonstrates that removal of one or both of the first two N-linkedglycosylation sites in soluble P-selectin ligand protein does notsignificantly effect its binding to P-selectin.

D. Effects of Tyrosines

Constructs were made to examine the role of tyrosine in P-selectinligand protein binding to selectins by alteration of the anionic regionof the soluble protein. The following constructs were made:

-   -   ΔY148.Fc amino acids 42-189 of SEQ ID NO:2, with amino acids        46-52 deleted    -   FFFE.148.Fc amino acids 42-189 of SEQ ID NO:2, with the tyrosine        residues at positions 46, 48 and 51 replaced with phenylalanine        residues and the aspartic acid residue at position 52 replaced        with a glutamic acid residue        These constructs are schematically represented in FIG. 8.

The degree and sites of sulfation of P-selectin ligand protein wereexamined by expressing relevant constructs in the presence ofradioactively labelled sulfate. The degree of sulfation of 148.Fc andΔY.148.Fc were compared to that of a P-selectin-IgG chimera, which wasnot sulfated. Results are depicted in FIG. 9. These data demonstratethat the majority of sulfate incorporation is into the anionic region ofthe P-selectin ligand protein.

Additional constructs were made to determine whether the sulfation ofthe anionic region occurred at the tyrosine residues. The followingadditional constructs were made:

-   -   FYYD.19.Fc amino acids 42-60 of SEQ ID NO:2, with the tyrosine        residue at position 46 of SEQ ID NO:2 replaced with a        phenylalanine residue    -   FFYD.19.Fc amino acids 42-60 of SEQ ID NO:2, with the tyrosine        residues at positions 46 and 48 of SEQ ID NO:2 replaced with a        phenylalanine residues    -   FFFD.19.Fc amino acids 42-60 of SEQ ID NO:2, with the tyrosine        residues at positions 46, 48 and 51 of SEQ ID NO:2 replaced with        phenylalanine residues        These constructs are schematically represented in FIG. 9.

The degree of sulfation of these constructs was compared to 19.Fc(“YYYD.19.Fc”). Results are shown in FIG. 10. FYYD.19.Fc showedsignificant sulfation while FFFD.19.Fc was substantially less sulfated.Thus, the tyrosine residues of the anionic region of P-selectin ligandproteins are the major site of sulfation.

Removal of sulfate from P-selectin ligand protein substantially reducesits binding to P-selectin. The binding of 148.Fc treated with chlorateto P-selectin was examined. As shown in FIG. 11, inhibition of sulfationby chlorate treatment substantially reduced the amount of P-selectinligand protein binding to P-selectin.

E. Effects of C-Terminal Deletions

Several additional C-terminal deleted constructs were made as follows:

254.Fc amino acids 42-295 of SEQ ID NO:2

47.Fc amino acids 42-88 of SEQ ID NO:2

19.Fc amino acids 42-60 of SEQ ID NO:2

These constructs are schematically represented in FIG. 12.

The binding of 254.Fc, 148.Fc, 47.Fc and 19.Fc to P-selectin, E-selectinand L-selectin was tested. FIGS. 23 and 24 compare the binding of thesedeletion chimeras to selectins and controls. Results are also summarizedin FIG. 12.

F. Binding to P-Selectin and E-Selectin Expressing Cells

Binding of various constructs described above to cells expressingP-selectin and E-selectin was compared using a quantitative platebinding assay of Example 4(c) (which is schematically described in FIG.13).

FIG. 14 compares the binding of 148.Fc, ΔY.148.Fc, FFFE.148.Fc and humanIgG1 to P-selectin expressing CHO cells. Deletion of all of the tyrosineresidues in the anionic region in ΔY.148.Fc eliminated binding. Changingthe tyrosine residues to phenylalanine residues in FFFE.148.Fcsubstantially reduced binding as compared to 148.Fc. Thus, it wasdemonstrated that the presence of the full length anionic region isessential to P-selectin binding and that P-selectin binding is enhancedby sulfation in this region. FIG. 14 also reports control experimentsdemonstrating that 148.Fc, ΔY.148.Fc and FFFE.148.Fc do not bind to CHOcells which do not express selectin.

FIG. 15 compares the binding of 148.Fc, ΔY.148.Fc, FFFE.148.Fc and humanIgG1 to E-selectin expressing CHO cells. E-selectin binding wasunaffected by the deletions or alterations of the native sequence. Thus,it was demonstrated that the anionic region is not required forE-selectin binding.

FIG. 16 summarizes the results of FIGS. 14 and 15.

FIG. 17 compares the binding of 47.Fc to P- and E-selectin expressingCHO cells. 47.Fc demonstrated substantial binding to both selectinsdespite deletion of the N-linked glycosylation sites at positions 111and 292 of SEQ ID NO:2.

FIG. 19 compares the binding of FYYD.19.Fc, FFFD.19.Fc, H24.Q70.148.Fc,148.Fc, and human IgG1 to P-selectin expressing CHO cells. Replacementof all of the tyrosine residues in the anionic region in FFFD.19.Fceliminated binding. Changing the tyrosine residue at position 46 to aphenylalanine residue in FYYD.19.Fc substantially reduced binding ascompared to 148.Fc. Alteration of the N-linked glycosylation sites inH24.Q70.148.Fc did not affect binding. Thus, it was demonstrated thatP-selectin binding is enhanced by sulfation in the anionic region andthat N-linked glycosylation is not required for P-selectin binding. FIG.19 also reports control experiments demonstrating that FYYD.19.Fc,FFFD.19.Fc, H24.Q70.148.Fc and 148.Fc do not bind to CHO cells which donot express selectin more than human IgG1 alone.

FIG. 20 compares the binding of FYYD.19.Fc, FFFD.19.Fc, H24.Q70.148.Fc,148.Fc, and human IgG1 to E-selectin expressing CHO cells. Truncation ofthe ligand protein to the degree of FYYD.19.Fc and FFFD.19.Fcsubstantially reduced E-selectin binding. Alteration of the N-linkedglycosylation sites in H24.Q70.148.Fc did not significantly affectE-selectin binding. Thus, it was demonstrated that P-selectin ligandproteins comprising amino acids 42 to 60 of SEQ ID NO:2 can selectivelybind P-selectin, and, to a substantially less, extent E-selectin.

FIG. 21 summarizes the results of FIGS. 19 and 20.

G. Conclusions Regarding P- and E-Selectin Binding

Although applicants do not which to be bound by any theory, these dataallow several conclusion regarding the relationship between P-selectinbinding and E-selectin binding by P-selectin ligand proteins. N-linkedcarbohydrates are not required for binding of a P-selectin ligandprotein to either P- or E-selectin. P-selectin ligand proteins as smallcomprising as little as amino acids 42-60 of SEQ ID NO:2 are capable ofbinding to P-selectin, and, to a substantially less, extent E-selectin.

FIG. 22 depicts a proposed schematic model for binding of P-selectinligand proteins to P- and E-selectin. O-linked sLe^(x) carbohydrate hasbeen demonstrated to be required for both P- and E-selectin binding.Data presented herein demonstrate that sulfated tyrosine residues areimplicated in P-selectin binding, but not E-selectin binding.Applicants' data also suggests that no N-linked glycosylation bindingsite is required.

EXAMPLE 11 Examination of Aggregation Phenomena and Dimer Formation inForms of PSGL-1

A panel of PSGL-1 mutants were constructed by site-directed mutagenesisand/or PCR amplification with primers that introduced a stopcodon. Thetemplate for all mutagenesis experiments was pPL85.R16 (ATCC 75577,deposited by applicants).

The first group of mutants (C310S and C327S) encode full-lengthPSGL-1.R16 with only one amino acid change compared to wild-typePSGL-1.R16 (Cys to Ser at position 310 or 327, respectively). COS cells,co-transfected with pEA.3/4FT and the mutants C310S or C327S, werelabeled with ³⁵S-methionine. Cell lysates were prepared and the mutantproteins were immunoprecipitated with the P-selectin ligand polyclonalantibody of Example 7(A) and analyzed by SDS-polyacrylamide gelelectrophoresis under non-reducing and reducing conditions.

The mutant C327S as well as wild-type PSGL-1.R16 migrated as a homodimerunder non-reducing conditions and as a monomer under reducingconditions. In contrast, the mutant C310S migrated as a monomer bothunder non-reducing and reducing conditions, indicating that the cysteineat position 310 is required for dimer formation of PSGL-1.

Both mutants were also analyzed for their ability to bind to P-selectin.Detergent extracts of co-transfected COS cells were precipitated withthe LEC-γ1 chimera of Example 4(A). The precipitates were analyzed bySDS-PAGE under non-reducing and reducing conditions and byautoradiography. Both PSGL-1.R16 and C327S were efficiently precipitatedby LEC-g1, whereas C310S binding to LEC-γ1 was greatly reduced,indicating that the dimeric form of PSGL-1 binds P-selectin more tightlythan the monomeric form.

The second set of mutants encode soluble forms of PSGL-1.R16 and arelisted in Table I. The mutant ΔTM was generated by site-directedmutagenesis and has a deletion of the transmembrane domain (amino acids313-333) followed by RLSRKA. The mutants L311, L312, A313, I314, L315,A318 and T322 were generated by site-directed mutagenesis or PCRamplication with PCR primers that introduced a stop codon in the desiredposition. The name of the mutant refers to the C-terminal amino acid ofeach truncated soluble form of PSGL-1.R 6. The mutants were analyzedaccording to the following criteria:

1. Expression and secretion from transfected COS cells

2. Dimer versus monomer formation

3. Lack of aggregate formation

4. P-selectin binding (LEC-γ1 chimera)

The mutants ΔTM and 1316 fulfilled all four criteria. The shortersoluble forms of PSGL-1, such as sPSL.QC of Example 5(A), L311, L312,A313, I314 and L315 did not form dimers as well and the longer solubleforms of PSGL-1, such as sPSL.T7 of Example 5(C), A318 and T322 formedhigh molecular weight aggregates which were less desirable.

CHO cells, already expressing 3/4 fucosyltransferase and Core2transferase, were transfected with psPSL.T7, ΔTM, I316 or psPSL.QC andamplified using methotrexate. Stable clones were isolated and labeledwith ³⁵S-methionine. Conditioned media was either analyzed directly orfirst precipitated with LEC-γ1 and then analyzed by SDS-PAGE undernon-reducing and reducing conditions (FIG. 25). The results indicatedthat ΔTM and I316 were most efficient in dimer formation and P-selectinbinding.

TABLE I Dimer High MW P-selectin Mutant Formation Aggregates bindingPSL.QC + − + L311 + − + L312 − − − A313 − − − I314 − − − L315 + − + I316++ − ++ A318 ++ + ++ T322 ++ + ++ ΔTM ++ − ++

EXAMPLE 12 Specificity of PSGL-1 Binding to P- and E-Selectins

Materials. A chimeric protein comprising the extracellular domain ofhuman E-selectin and the Fc portion of human IgG₁ was constructedanalogously to the P-selectin chimera, LEC-γ1, described earlier. Thesoluble E-selectin chimera was expressed in baculovirus-infectedTrichoplusia ni high five cells (Invitrogen) and purified to homogeneityby Protein A Sepharose chromatography. Plasmid vectors pEA.3/4FT, pPL85,pFCD43, and pEA.sPACE, for COS expression ofa(1,3/1,4)-fucosyltransferase (Fuc-TIII), PSGL-1, CD43 (leukosialin),and soluble paired basic amino acid converting enzyme (PACE),respectively, have been described herein and in the literature (Sako etal. (1993) Cell 75, 1179-1186; Rehemtulla, A. & Kaufman, R. J. (1992)Curr. Opin. Biotechnol. 3, 560-565; Wasley et al. (1993) J. Biol. Chem.268, 8458-8465). Fuc-TVII cDNA (plasmid pMT.FT7) was cloned from an HL60cDNA expression library using oligonucleotide probes derived from thepublished sequence (Natsuka et al. (1994) Journal of BiologicalChemistry 269, 16789-16794; Sasaki et al. (1994) J. Biol. Chem. 269,14730-14737). A polyclonal neutralizing rabbit antibody, Rb3443, wasraised against a peptide comprising the first 15 amino acids of themature (PACE-cleaved) N-terminus of PSGL-1. Monoclonal anti-CD43antibodies from either Becton Dickinson or Biodesign International andisotype control antibodies were coupled to a solid support consisting ofSepharose TM-4B with a covalently attached goat affinity-purifiedantibody to mouse IgG (Cappel, Organon Teknika Corporation). Affinitycoupling of selectin chimeras and murine antibodies to Protein ASepharose 4 Fast Flow (Pharmacia) and to the anti-mouse IgG resin,respectively, was carried out at a ratio of 2 mg protein/ml of resin.Antiserum Rb3443 was coupled to Protein A Sepharose at 1 ml/ml resin.Coupling efficiencies, indicated by micro-BCA assay (Pierce) of thepost-reacted supernatants, were at least 95%. Aprotinin and pepstatinwere from Boehringer Mannheim and benzamidine, leupeptin, andphenylmethylsulfonyl fluoride (PMSF) were from Sigma.

Labeling and Membrane Extraction of Myeloid Cells. U937 or HL60 cellsgrown in suspension to a density of ˜1.3×10⁶ cells/ml were labeled in 50ml of RPMI1640 medium supplemented with 10% fetal bovine serum and 2.5mCi of ³H-glucosamineHCl (Dupont/NEN) for 48 hr. Activities of greaterthan 1 cpm/cell were routinely obtained by this technique. The labeledcells were washed with PBS, resuspended in cell lysis buffer (10 mMMOPS, 150 mM NaCl, 4 mM CaCl₂ and 4 mM MgCl₂, pH 7.5 containing proteaseinhibitors 20 mg/ml aprotinin, 10 mM benzamidine, 20 mg/ml leupeptin, 8mg/ml pepstatin, and 10 mM PMSF) and subjected to several cycles ofprobe sonication on ice. Nuclei and cell debris were removed by lowspeed centrifugation and the cell membranes recovered from thesupernatant by centrifugation at 100,000 g RCF for 1 hr, washed byresuspension and high speed centrifugation in cell lysis buffercontaining 1 M NaCl, and finally resuspended in 3 ml membranesolubilization buffer (cell lysis buffer containing 1% Triton X-100).Several cycles of sonication and incubation on ice were employed tosolubilize the membrane fraction. Finally, a low speed centrifugationstep was employed to remove insoluble membrane residue.

Labeling and Membrane Extraction of Transfected COS Cells. COS M6 cellswere transfected using DEAE-dextran and chloroquine (25) employing 8 μgof plasmids pPL85 or pFCD43 and 4 μg of pEA.sPACE, as well as 4 μg ofpEA.3/4FT or pMT.FT7. After 40-45 hr recovery the transfected cells werestarved in serum- and methionine-free DME medium for 30 min and then fed[³⁵S]-methionine in serum-free DME for 5 hr. The labeled cells werewashed, incubated with EGTA to loosen them from the dish surface,scraped from the dish, pelleted, and suspended in cold 10 mM PIPESbuffer, pH 7.5, containing 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl₂, andprotease inhibitors (see above). Membrane extraction then was carriedout by sonication, low speed centrifugation, high speed centrifugation,and solubilization in membrane lysis buffer as above, for labeledmyeloid cells.

Affinity Precipitations. Membrane extracts were diluted 1:4 or 1:5 withcell lysis buffer or with TBSC buffer (20 mM Tris HCl, 150 mM NaCl, 2 mMCaCl₂, pH 7.5) supplemented with 5 mg/ml bovine serum albumin(approximately 99%, Sigma). Extracts thus diluted to 0.2-0.25% TritonX-100 were incubated with human IgG₁-Protein A Sepharose withend-over-end mixing at 4° C. overnight. The precleared supernatants thenwere reacted for 6-12 hrs at 4° C. with Protein A Sepharose precoupledwith E- or P-selectin chimeras, control human IgG₁, Rb3443 or withrabbit pre-immune serum or with anti-CD43 antibody or isotype controlprecoupled to goat anti-mouse IgG Sepharose. The resins were washed 5 ormore times in buffer containing 0.1-0.5% Triton X-100 until theradioactivity of the wash supernatants was reduced to background level.Elution of proteins bound specifically to P- or E-selectin resins wasaccomplished with 10 mM EDTA or 5 mM EDTA/5 mM EGTA at room temperatureor by boiling in SDS-PAGE sample buffer (Laemmli, U. K. (1970) Nature227, 680-685), whereas elution of proteins bound to antibody resins wasachieved exclusively by the latter means. For resolution under reducingconditions, dithiothreitol was added to the sample buffer to a finalconcentration of 100 mM. Samples thus prepared were resolved by SDS-PAGEon 7.5% gels, treated with En³Hance (Dupont), dried, and exposed toautoradiography film.

For sequential affinity capture experiments, membrane extracts wereprecleared, affinity precipitated with P- or E-selectin or human IgG₁,and washed as above. Samples then were eluted twice from the resins with5 mM EDTA in 10 mM MOPS, 150 mM NaCl, pH 7.5 for 1 hr at 4° C. withtumbling. The first and second eluates were combined and thenimmunoprecipitated with immobilized Rb3443 according to the protocolsoutlined above.

Results:

Soluble E- and P-selectin chimeras were used, in parallel with controlhuman IgG₁ to probe detergent-solubilized membrane extracts of³H-glucosamine-labeled U937 cells as described under “Methods”.Examination of eluates from the immobilized selectins bySDS-PAGE/autoradiography (FIG. 26) revealed the presence in both P- andE-selectin eluates of a major protein species with identicalelectrophoretic properties: Mr 200-kDa non-reduced with conversion to aspecies of Mr 120-kDa following reduction (FIG. 26, lanes 2 and 3,respectively). Occasionally, additional bands were observed in both E-and P-selectin eluates presumably corresponding to this major band andreflecting the presence of naturally reduced material (the 120-kDaspecies in non-reduced samples) and incomplete reduction (the 200-kDaspecies in reduced samples). Additionally, a trace band of Mr 150-kDa inthe E-selectin eluate which was unaffected by reduction with DTT wasoccasionally observed. No bands were observed in control experimentsusing immobilized human IgG₁ (FIG. 26, lane 1) or where elution ofselectin resins was performed in the absence of EDTA or SDS (data notshown). Essentially identical results were obtained using HL-60 cells(data not shown). Hence, the nature of these recognition events isinterpreted to be specific metal-dependant interactions of theseproteins with the respective selecting, presumably via the lectindomains (Lasky, L. A. (1992) Science 258, 964-969; Drickamer, K. (1988)J. Biol. Chem. 263, 9557).

The metal-dependant recognition and electrophoretic behavior of themajor band precipitated with E-selectin was consistent with theproperties of the previously identified P-selectin counterreceptor,P-selectin glycoprotein ligand or PSGL-1 (Moore et al. (1994) J. Biol.Chem. 269, 23318-23327; Moore et al. (1992) J. Cell Biol. 118, 445-456;Sako et al.).

To assess whether this species was indeed PSGL-1, EDTA eluates of theboth E- and P-selectin precipitates were subsequently reacted with thePSGL-1 specific polyclonal antiserum Rb3443. As shown in FIG. 27, themajor band isolated by affinity capture with either selectin wasimmunoprecipitated using this antiserum (lanes 3 and 4, respectively).No species were detected after immunoprecipitation of the control IgG₁EDTA eluate (FIG. 27, lane 5). Direct immunoprecipitations using fresh³H-labeled U937 membrane extracts confirmed the specificity of Rb3443:precipitation with Rb3443 results in the recovery of a single band withthe electrophoretic properties of PSGL-1 (Mr 200-kDa non-reduced, Mr120-kDa reduced; FIG. 27, lane 2) whereas precipitation with pre-immuneantiserum fails to capture any material (FIG. 27, lane 1). These resultsindicate that the major protein species specifically captured frommyeloid cells by both E- and P-selectins is PSGL-1.

To further assess the specificity of E-selectin for PSGL-1, U937membrane lysates were probed directly for the presence of CD43 (orleukosialin), an abundant cell surface sialoglycoprotein known to bearthe major portion of myeloid cell SLe^(X) residues (Maemura, K. &Fukuda, M. (1992) J. Biol. Chem. 267, 24379-24386). Thus, membraneextracts of ³H-glucosamine-labeled U937 cells were probed with ananti-CD43 antibody in parallel with the PSGL-1 specific antiserum Rb3443and control antibodies as described under “Methods”. From identicalquantities of membrane lysate, the CD43 antibody precipitated in excessof 30-fold greater radioactive counts than did the PSGL-1 antiserum.Evaluation of the immunoprecipitates by SDS-PAGE/autoradiography (FIG.28) revealed a single specific band for each antibody. Rb3443 captured asingle species with the electrophoretic characteristics of PSGL-1 (FIG.28, Lane 2). In contrast, the CD43 antibody precipitated a species withan Mr 120-kDa which was insensitive to reduction (FIG. 28, Lane 4),consistent with the absence of cysteine in CD43. Immunoprecipitationswith control antibodies (FIG. 28, lanes 1 and 3) proved negative asexpected. There appears to be considerably greater quantities of CD43than PSGL-1 in U937 cells, consistent with the quantitation of theseproteins in HL-60 cells (Ushiyama et al. (1993) J. Biol. Chem. 268,15229-15237). Thus, the inability of E-selectin to precipitate CD43 frommyeloid cells does not appear to be due to its absence in these celllines. While we cannot exclude the possibility that E-selectin capturestrace quantities of CD43 (ie., the low-intensity Mr 120-kDa band in FIG.26, non-reduced lane 3 which is also consistent with monomeric PSGL-1),PSGL-1 appears to be the major protein precipitated from myeloidmembrane extracts.

Recombinant PSGL-1 expressed in COS cells is best achieved withcotransfection of the PSGL-1 cDNA with a cDNA encoding an a(1,3/1,4)fucosyltransferase (Fuc-TIII) for P-selectin binding (Sako et al.).Interestingly, initial efforts to demonstrate E-selectin recognition ofrecombinant PSGL-1 failed: E-selectin was unable to capture thecounterreceptor from cotransfected COS cell membrane lysates underconditions where P-selectin capture was successful. One interpretationof this result is that Fuc-TIII was able to modify recombinant PSGL-1for recognition by P-selectin but was unable to replicate theappropriate modification(s) found in myeloid PSGL-1 necessary forE-selectin recognition. The recent cloning of a myeloidfucosyltransferase, Fuc-TVII (Natsuka et al. (1994) Journal ofBiological Chemistry 269, 16789-16794; Sasaki et al. (1994) J. Biol.Chem. 269, 14730-14737), that is also capable of generating SLe^(x)carbohydrate structures, allowed evaluation of this interpretation.

COS cells were cotransfected with cDNAs encoding either PSGL-1 or CD43and either Fuc-TIII or Fuc-TVII. Membrane lysates were prepared from thetransfected COS cells and these were precipitated with eitherimmobilized E- or P-selectin chimeras or with antibodies to eitherPSGL-1 or to CD43. The precipitated products were evaluated bySDS-PAGE/autoradiography following their release by EDTA/EGTA (forselectin mediated binding) or by boiling in SDS (forimmunoprecipitations). The results are shown in FIG. 29.

As observed in FIG. 29A, E-selectin capture of COS-expressed PSGL-1 wasdependant upon the nature of the fucosyltransferase used in thetransfection. In three separate experiments, Fuc-TVII, but not Fuc-TIII,supported PSGL-1 precipitation by E-selectin. The inability of Fuc-TIIIto confer E-selectin reactivity to PSGL-1 cannot be attributed to a lackof PSGL-1 expression as the specific antiserum Rb3443 immunoprecipitatedsignificant and comparable quantities of PSGL-1 from both Fuc-TIII andFuc-TVII transfections (FIG. 29C). Furthermore, P-selectin was capableof precipitating equivalent quantities of PSGL-1 with eitherfucosyltransferase (FIG. 29B), demonstrating that Fuc-TIII and Fuc-TVIIare expressed and active in these cotransfections.

Within the COS recombinant expression system as in myeloid cells,high-affinity E-selectin recognition was also dependent upon thepresence of an appropriate polypeptide. Although the polypeptide length,apparent molecular weight, and high frequency and specific types ofposttranslational modifications are similar in CD43 and PSGL-1 (Maemura,K. & Fukuda, M. (1992) J. Biol. Chem. 267, 24379-24386), neitherfucosyltransferase was able to confer high-affinity E-selectin (orP-selectin) recognition to recombinant leukosialin in cotransfected COScells (FIG. 29A). Immunoprecipitations with the anti-CD43 antibodyindicate that comparable quantities of leukosialin were expressed inboth Fuc-TIII and Fuc-TVII cotransfections (FIG. 29C). The failure ofE-selectin to capture CD43 was not due to lack of fucosyltransferaseactivity within the cotransfected COS cells. FACS analysis of COS cellstransfected with either PSGL-1 or CD43 and either Fuc-TIII or Fuc-TVIIall show high levels of reactivity with the SLeX specific antibodyCSLEX-1. Therefore, these results suggest that high-affinity E-selectinrecognition requires the presence of a specific polypeptide(s) that isappropriately modified by a specific fucosyltransferase.

EXAMPLE 13 Inhibition of P-Selectin/PSGL-1 Binding by PSGL-1 DerivedPeptides

A number of peptides derived from the sequence of PSGL-1 (SEQ ID NO:2)were tested for their ability to inhibit P-selectin/PSGL-1 binding. Thetested peptides are listed in FIG. 30.

Inhibition was tested according to the following protocol. The wells ofa 96 well plate were coated overnight at 4° C. with PSGL-1 in 50 μl of10 mM MOPS, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl at pH 7.5. After removalof the liquid from the wells, 150 μl of 10 mM MOPS, 150 mM NaCl, 1 mMCaCl₂, 1 mM MgCl₂, 0.05% tween-20, 0.05% gelatin at pH 7.5 was added perwell to block the unoccupied sites. After ½ hr. to 2 hr. the blockbuffer was removed from the wells and 100 μl of a complex of Lec-γ1(P-selectin-human IgG Fc chimera) (2 μg/ml), biotinylated goatanti-human antibody, and streptavidin-conjugated alkaline phosphatase(which had been allowed to tumble at room temperature for 30 minutes to1 hour) plus any potential inhibitors were added per well. Each platewas shaken and rapped to remove the block from plate. The incubationproceeded for 1 hr at room temperature rotating in the dark. The unboundcomplex was washed off the plate with two 150 μl portions of 10 mM MOPS,150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 0.05% tween-20 followed by 150 μlof 1M diethanolamine, 0.5 mM MgCl₂. The chromogenic substrate foralkaline phosphatase, PNPP, in 10 mM DEA/0.5 mM MgCl₂ was added and theplate is then read at 405 nm.

The results of these assays are reported in FIG. 30. The peptidescomprising amino acids 48-51 (in which the tyrosine residues have beenphosphorylated) and amino acids 42-56 of SEQ ID NO:2 providedparticularly desirable results.

EXAMPLE 14 Purification of a Soluble Form of PSGL-1

Substantial purification of a soluble form of P-selectin ligand proteinhas been achieved according to the protocol described below.

A soluble P-selectin ligand protein, 1316 (amino acid 42 to amino acid316 of SEQ ID NO:2) was expressed in CHO cells as described herein. CHOcell conditioned media was concentrated with a Pellicon ultrafiltrationmembrane unit (Millipore) with either 10,000 molecular weight cutoff(MWCO) or 30,000 MWCO to about 10 times the original concentration. Thebuffer was then exchanged into 25 mM Tris, 1 mM CaCl₂, pH 7.4.

The buffer-exchanged concentrate was loaded onto a Toyopearl QAE 550C(TosoHaas) column. Alternatively, the buffer exchange step can beomitted and the concentrate can be diluted one part concentrate to threeparts 25 mM Tris, 1 mM CaCl₂, pH 7.4, and then loaded onto the column.The column was washed with 5-10 column volumes (CV) of 25 mM Tris, 1 mMCaCl₂, pH 7.4 at 4° C.

The P-selectin ligand protein eluted from the column with a linear NaClgradient (0 M NaCl to 1.0 M NaCl) in the 25 mM Tris, 1 mM CaCl₂, pH 7.4buffer in approximately five column volumes. Two peaks were eluted fromthe column. The second peak contained the P-selectin ligand protein andwas collected in bulk.

The peak from the QAE column was concentrated with a tangential flowultrafiltration membrane (Millipore) with a 30,000 MWCO and was thenbuffer exchanged into 25 mM Tris, 150 mM NaCl, 1 mM CaCl₂, pH 7.4 at 4°C.

The buffer exchanged concentrate was loaded onto a Jacalin Agarosecolumn overnight at 4° C. The column was washed with the diafiltrationbuffer and the P-selectin ligand protein was eluted with a gradient ofmethyl α-D-galactopyranoside (0-100 mM or)-50 mM methylα-D-galactopyranoside) at 20° C. Fractions from the Jacalin column wereanalyzed by SDS-PAGE and the purest fractions were pooled.

EXAMPLE 15 P-Selectin Ligand Protein Fusions

Four fusions of a P-selectin ligand protein with a different amino acidsequence were constructed: 47.Fc, 47.AGP, 47.BMP and 47.IL11.

47.Fc: A cDNA was constructed encoding the signal peptide, PACE cleavagesite and first 47 amino acids of the mature P-selectin ligand sequencefused to a mutated Fc region of human IgG1 at His224 of the native Fcsequence. The sequence of the cDNA construct is reported as SEQ IDNO:35. The fusion point is a novel NotI site at nucleotide 261. Theamino acid sequence encoded by the cDNA construct is reported as SEQ IDNO:36. The mature amino acid sequence of the encoded fusion proteinbegins at amino acid 42 of SEQ ID NO:36. The mutations in the Fc portionwere a change of Leu 234 and Gly237 of the native Fc sequence to Ala.

47.AGP: A cDNA was constructed encoding the signal peptide, PACEcleavage site and first 47 amino acids of the mature P-selectin ligandsequence fused to the first leucine residue of mature human AGP. Thesequence of the EDNA construct is reported as SEQ ID NO:37. The fusionpoint is a novel NotI site at nucleotide 261. The amino acid sequenceencoded by the cDNA construct is reported as SEQ ID NO:38. The matureamino acid sequence of the encoded fusion protein begins at amino acid42 of SEQ ID NO:38.

47.BMP: A cDNA was constructed encoding the signal peptide, PACEcleavage site and first 47 amino acids of the mature P-selectin ligandsequence fused to the sequence of mature human BMP-2 (with its first 8amino acids deleted). The sequence of the cDNA construct is reported asSEQ ID NO:39. The fusion point is a novel NotI site at nucleotide 261.The amino acid sequence encoded by the cDNA construct is reported as SEQID NO:40. The mature amino acid sequence of the encoded fusion proteinbegins at amino acid 42 of SEQ ID NO:40.

47.IL11: A cDNA was constructed encoding the signal peptide, PACEcleavage site and first 47 amino acids of the mature P-selectin ligandsequence fused to mature human IL-11. The sequence of the cDNA constructis reported as SEQ ID NO:41. The fusion point is a novel NotI site atnucleotide 261. The amino acid sequence encoded by the cDNA construct isreported as SEQ ID NO:42. The mature amino acid sequence of the encodedfusion protein begins at amino acid 42 of SEQ ID NO:42.

Patent and literature references cited herein are incorporated as iffully set forth.

1. An isolated DNA encoding a fusion protein comprising (a) a firstamino acid sequence comprising amino acid 42 to amino acid 60 of SEQ IDNO:2, and (b) a second amino acid sequence derived from the sequence ofa protein other than P selectin ligand.
 2. The DNA of claim 1 whichfurther comprises an expression control sequence operably linked to saidnucleotide sequence.
 3. An isolated host cell transformed with the DNAof claim
 2. 4. A process for producing a fusion protein, whichcomprises: (a) culturing the host cell of claim 3 under conditionsuitable for expression of the fusion protein; and (b) purifying thefusion protein from the culture medium.
 5. The DNA of claim 1 whereinsaid first amino acid sequence comprises amino acid 42 to amino acid 402of SEQ ID NO:2.
 6. The DNA of claim 1 wherein said first amino acidsequence comprises amino acid 42 to amino acid 310 of SEQ ID NO:2. 7.The DNA of claim 1 wherein said first amino acid sequence comprisesamino acid 42 to amino acid 88 of SEQ ID NO:2.
 8. The DNA of claim 1wherein said first amino acid sequence comprises amino acid 42 to aminoacid 118 of SEQ ID NO:2.
 9. The DNA of claim 1 wherein said first aminoacid sequence comprises amino acid 42 to amino acid 189 of SEQ ID NO:2.10. The DNA of claim 1 wherein said second amino acid sequence is linkedto the C-terminus of said first amino acid sequence.
 11. The DNA ofclaim 10 wherein said sequences are linked by a linking sequence. 12.The DNA of claim 1 wherein said second amino acid sequence is joined tothe N- terminus of said first amino acid sequence.
 13. The DNA of claim12 wherein said sequences are linked by a linking sequence.
 14. The DNAof claim 1 wherein said second amino acid sequence is an Fc portion ofan antibody.
 15. The DNA of claim 14, wherein the Fc portion of anantibody is from a human antibody.
 16. The DNA of claim 15, whereinLeu234 and Gly237 of the Fc portion of the human antibody are changed toAla.